Download - Pathology Research 2013 - wako-chem.co.jp

Pathology Research 2013A. Fixatives 3

A-1. for Light Microscopy .......................................................................................3A-2. for Electron Microscopy .................................................................................5

B. Tissue Dehydrating Solution 6

C. Decalcifying Agent 7

D. Intermediates (Replacing agents) 9

D-1. Lemosol® and Lemosol® A (Ace) ................................................................9

E. Embedding Medium 11

F. Immunopen 12

G. Stains 13

G-1. General Staining .............................................................................................14G-2. Polysaccharide Staining ..............................................................................16G-3. Connective Tissue Staining ........................................................................19G-4. Staining of Fibrins and Glial Tissues .......................................................23G-5. Intracellular Inorganic Substance Staining ...........................................23G-6. Cytological Staining ......................................................................................24G-7. Blood Cell Staining ........................................................................................25G-8. Hard Tissue Staining (TRAP/ALP Stain Kit) ........................................26G-9. Other stains ......................................................................................................27

H. Mounting Reagent 28

H-1. Softmount ..........................................................................................................28

I. Antigen Retrieval Reagent 28

I-1. ImmunoSaver ...................................................................................................28

J. Gene-Related Kits 29

J-1. Apoptosis in situ Detection Kit Wako ......................................................... 29J-2. DNA Isolator PS Kit ....................................................................................... 30J-3. DNA Isolator PS-Rapid Reagent ............................................................. 30J-4. ISOGEN PB Kit ................................................................................................31

K. Paraffin-embedded Tissue Sections 32

K-1. HistoMap Series ............................................................................................. 32

Alphabetical Indexpage Description

27 A Acridine Orange27 Acridine Yellow17 Alcian Blue Solution, pH 2.522 Aniline Blue · Orange G Solution21 Aniline Blue Solution27 Aniline Blue · Water Soluble28 Apathy's Mounting Media, Soluble29 Apoptosis in situ Detection Kit Wako29 Apoptosis Ladder Detection Kit Wako27 Auramine22 Azocarmine G Solution23 B Berlin Blue Staining Set27 Bismarck Brown5 Bouin Solution28 C Canada Balsam27 Carmine5 Carnoy Solution15 Carrazzi's Hematoxylin Solution15 Carrazzi's Hematoxylin Solution (×2)27 Chlorazol Black E16 Cold Schiff’s Reagent11 Collodion (5%)11 Collodion (10%)19 Colloidal Iron Stock Solution27 Congo Red27 Crystal Violet29 D DAB Tablet (DAB . 4HCl 5mg/Tablet)29 DAB Tablet (DAB . 4HCl 10mg/Tablet)29 DAB TRIS Tablet, pH7.68 Decalcifying Solution A (Plank-Rychlo protocol)8 Decalcifying Solution B (EDTA protocol)27 4',6-Diamidino-2-phenylindole Dihydrochloride n-Hydrate30 DNA Isolator PS Kit30 DNA Isolator PS-Rapid Reagent11 Dodecenylsuccinic Anhydride [DDSA]15 E Eosin Alcohol Solution, acid extract27 Eosin Y15 0.1% Eosin Y Ethanol Solution15 0.5% Eosin Y Ethanol Solution15 1% Eosin Y Solution11 Epon 17411 Epon 81211 Epon 81527 Erythrosine30 Ethanol (99.5)11 Ethylene Glycol Diglycidyl Ether27 F Fast Blue RR Salt3 10% Formalin Neutral Buffer Solution3 15% Formalin Neutral Buffer Solution3 20% Formalin Neutral Buffer Solution8 5% Formic Acid (for Decalcification)27 Fuchsin Acid27 Fuchsine Basic11 G Gelatin11 Gelatin, Capsules No.011 Gelatin, Capsules No.0027 Gentian Violet R25 Giemsa Stain Solution6 10% Glutaraldehyde Solution6 20% Glutaraldehyde Solution6 70% Glutaraldehyde Solution27 H Hematoxylin Monohydrate32 HistoMap Mouse Normal Cerebellum, Paraffin Embedded Tissue32 HistoMap Mouse Normal Cerebrum, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Heart, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Kidney, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Liver, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Lung, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Pancreas, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Rectum, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Spleen, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Stomach, Paraffin Embedded Tissue Section32 HistoMap Mouse Normal Testis, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Cerebellum, Paraffin Embedded Tissue32 HistoMap Rat Normal Cerebrum, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Heart, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Kidney, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Liver, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Lung, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Pancreas, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Rectum, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Spleen, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Stomach, Paraffin Embedded Tissue Section32 HistoMap Rat Normal Testis, Paraffin Embedded Tissue Section8 10% Hydrochloric Acid (for Decalcification)

page Description12 I Immunopen28 ImmunoSaver27 Indigo Carmine31 ISOGEN-LS31 ISOGEN PB Kit7 K Kalkitox™

10 L Lemosol®10 Lemosol® A (Ace)10 Lemosol® Coagulant27 Light Green SF Yellowish27 M Malachite Green Oxalate14 Mayer’s Hematoxylin Solution14 Mayer’s Hematoxylin Solution (×2)25 May-Grünwald Stain Solution25 May-Grünwald's Eosin Methylene Blue Solution27 Metanil Yellow27 Methyl Green27 Methyl Violet B11 Methyl-5-norbornene-2,3-dicarboxylic Anhydride [MNA]27 Methylene Blue Tetrahydrate4 Mildform 10N4 Mildform 10NM4 Mildform 15N4 Mildform 15NM4 Mildform 20N4 Mildform 20NM8 Morse Solution

27 N Neutral Red27 Nile Blue Hydrogensulfate27 Nile Red8 8% Nitric Acid (for Decalcification)

11 Nonenylsuccinic Anhydride [NSA]27 O Oil Red O27 Orange G21 0.8% Orange G Solution27 Orcein6 Osmium (VIII) Oxide6 2% Osmium (VIII) Oxide Solution6 4% Osmium (VIII) Oxide Solution

24 P Papanicolaou EA100 Stain Solution24 Papanicolaou Hematoxylin Stain Solution24 Papanicolaou OG100 Stain Solution24 Papanicolaou-Gill's Hematoxylin (Gill-5) Stain Solution6 Paraformaldehyde5 4% Paraformaldehyde Phosphate Buffer Solution

11 Pathoprep® 54611 Pathoprep® 56811 Pathoprep® 58027 Phloxine B23 Phosphotungstic Acid Hematoxylin Solution5 PLP Solution Set

21 Ponceau Xylidine, Fuchsin Acid, Azophloxine Solution30 2-Propanol27 Pyronine Y27 S Safranine16 Schiff’s Reagent27 Sirius Red8 5% Sodium Sulfate Solution (for Decalcification)

28 Softmount27 Sudan 127 Sudan 227 Sudan 327 Sudan 427 Sudan Black B27 T Thioflavin T27 Thionin Acetate6 Tissue Dehydration Solution 996 Tissue Dehydration Solution 100

18 0.05% Toluidine Blue Solution (pH2.5)18 0.05% Toluidine Blue Solution (pH4.1)18 0.05% Toluidine Blue Solution (pH7.0)26 TRAP/ALP Stain Kit8 5% Trichloroacetic Acid Solution (for Decalcification)6 2,4,6-Trimethylpyridine [γ-Collidine]

11 2,4,6-Tris (dimethylaminomethyl) phenol [DMP-30]20 V Van Gieson Solution F20 Van Gieson Solution P27 Victoria Blue B27 Victoria Blue 4R22 Victoria Blue Solution19 W Weigert's Iron Hematoxylin Staining Set27 Wright Stain, Powder25 Wright Stain Solution5 Z Zamboni Solution

Pathology Research 2013 3

AFixatives

A. Fixatives

F ixation means a process by which when observing microscopically the ecological or pathological state of a tissue specimen taken from a dead or live body, the tissue specimen is preserved in as "intact" a state as possible.

The objectives are to:l Stabilize the main constituent protein of the tissue specimen as fast as possible to stop autolysisl �Minimize the degeneration and deformation of the tissue specimen caused by the effects of chemicals and heat during

the subsequent specimen preparation process.

A-1. for Light Microscopy

Neutral buffered formalin Formalin solution is used in large quantities as a routine fixative in the preparation of tissue specimens for light microscopic observation. Formalin is decomposed to produce formic acid, which may affect some tissues. This neutral buffered formalin is adjusted to pH 7.4 approximately by the addition of sodium phosphate to formalin to eliminate the effect of acid on tissues.Not only is neutral buffered formalin a superior fixative for routine lab work, it is also beneficial for a number of special purposes. For example, neutral buffered formalin can be used to a certain extent for histochemical detection of enzymes, hemoglobin and other molecules; for fixing mucopolysaccharides, glycogens, neurofibrils and other tissue components that cannot be treated with regular formalin; and for electron microscopy.

Product Name Composition Formaldehyde Content pH Penetration Strength

10% Formalin Neutral Buffer Solution

Formalin ......................................................................100 mLSodium phosphate, monobasic, Dihydrate ........4.5 gDisodium phosphate ...................................................6.5 gPrepare the 1 L solution to add ion-exchanged water

3.7~4.3 w/w% 7.4~7.5 l �Penetration strength is in the order of 20% > 15% > 10%.

l �Specimens must be placed in the 10% fixative for several days or more.

l �The 20% fixative allows fixation in one or two days.

l �The 15% solution is about halfway between the 10% and 20% solutions.


Formalin ......................................................................150 mLSodium phosphate, monobasic, Dihydrate ........4.5 gDisodium phosphate ...................................................6.5 gPrepare the 1 L solution to add ion-exchanged water

5.6~6.4 w/w% 7.35~7.45


Formalin ......................................................................200 mLSodium phosphate, monobasic, Dihydrate ......4.5 gDisodium phosphate ...................................................6.5 gPrepare the 1 L solution to add ion-exchanged water

7.5~8.5 w/w% 7.3~7.5

Product Name Grade Package Size Wako Cat. No.

10% Formalin Neutral Buffer Solution for Tissue Fixation1 L 062-01661

20 L 060-01667


20 L 067-02397


20 L 068-01727

Mildform Mildform products, fixatives for pathological tissues, are neutral buffered formalin solution prepared according to Lillie’s formulation to which wine extractk is added. The wine extract minimizes the irritating and unpleasant odor of formalin, however, with regard to operational safety, minimum degree of odor is present so that formalin can be recognized.k�Mechanism of wine extract’s effect: The masking effect minimizes the irritating and unpleasant odor of formalin.

Featuresl Most suitable for tissue fixation of immunohistochemical staining by an immunoenzymatic technique.l Mildform can preserve tissue in the advanced stages of autolysis without compromising visual clarity.l Stable for more than 2 years at room temperaturel �This solution, which lacks the unpleasant odor of conventional formalin, is useful for biopsies, experimental tissue

sections and surgical resections. It is also ideal for quick fixation of anatomical tissue in the operating room or pathology lab.

l The fixation and osmosis are equivalent to that of neutral buffered formalin solution or greater.

Pathology Research 2013 for other products, please visit the Wako Online Catalog www.e-reagent.com4

AFi

xativ

es

Mildform A-1. for Light Microscopy


Mildform 10N for Pathology Research1 L 133-10311

20 L 131-10317


20 L 130-14307


20 L 134-10047

Mildform 10NM for Pathology Research1 L 132-10521

20 L 130-10527


20 L 137-14317


20 L 137-10537

l N: signifying neutral. Mildforms are produced based on neutral buffered formalin.l M: signifying methanol content. Addition of methanol enhances osmosis into and fixation to tissues. Methanol is added to the product at 10%

amount of the package size.l �In general, NM-type is most suitable for rapid fixation with minimum length of time needed. Also suitable as a formalin-methanol fixative for fixing

samples containing a large amount of connective tissues and adipose tissues.<Note> Although Mildform lacks the unpleasant, acerbic odor of formalin, it is still as toxic as regular formalin and therefore should be handled with

due care.

Other Fixatives

Product Name Composition Features

Bouin SolutionSaturated Picric Acid Solution ..........................750 mLFormalin Stock Solution ......................................250 mLAcetic Acid, Glacial ................................................. 50 mL

This product allows fixation of small sections in 1 to 2 hours because of its strong penetration. Fetal bone tissue can be thinly sliced because the solution’s decalcifying effect is relatively weak. This fixative is used frequently for endocrine tissue, glycogens and polysaccharides. After fixation, wash specimen with 70% ethanol until the yellow color disappears.

Carnoy SolutionPure Ethanol .............................................................600 mLChloroform ................................................................300 mLAcetic Acid, Glacial ...............................................100 mL

With better penetration performance than anhydrous alcohol, this product is suited for the fixation of glycogens and nucleic acids. It can fix 3 to 4 mm specimens in a matter of 2 to 4 hours. After fixation, transfer the specimen to pure alcohol to remove chloroform and acetic acid, and then embed. Because this product has strong dehydrating and delipidating proper-ties, overfixation will lead to extreme contraction and hardening of the specimen.

4% Paraformaldehyde Phosphate Buffer

Solution

Paraformaldehyde .........................................20 g0.1 mol/L Phosphate buffer (pH 7.4) ...........500 mL

The product is used in enzyme histochemistry and immunohis-tochemistry, and as a fixing solution for electron microscopy.It is used for fixing protein antigens in immunostaining.

Data

Mildform 20N Fixation

Immunohistological staining of human lymph node tissue fixed with Mildform 20N(Source: Pathology Department, Osaka Koseinenkin Hospital)

1. Mildform 10N (Equivalent to 10% Formalin Neutral Buffer Soln.)2. Mildform 10NM3. Mildform 20N (Equivalent to 20% Formalin Neutral Buffer Soln.)4. Mildform 20NM5. 10% Formalin Soln.6. 20% Formalin Neutral Buffer Soln.

LCA

panB

20% Formalin Neutral Buffer Soln. Fixation Comparison of penetration/fixation rates for human liver tissue

(mm)

(time)

109

8

7

6

5

43

2

1

01 2 3 4 5 6 7 8 10 12 14 16 18 20 22 24

12

6

3

4

5

Pene

tratio

n/fix

atio

n


AFixatives

A-1. for Light MicroscopyOther Fixatives

Product Name Composition Features

PLP Solution Set

Solution A (Lysine-phosphate buffer solution) .................. 50 mL Solution B (10 % Paraformaldehyde solution) .................. 50 mLSolution C (Metaperiodic acid solution) .............................. 50 mL

This neutral buffered fixative is specially made for immunohis-tochemistry and is suited for fixing glycoprotein antigens. It is used to detect surface makers of complements and lympho-cytes that are prone to losing their antigenicity.

Zamboni Solution

Saturated Picric Acid Solution ..........................150 mLParaformaldehyde .................................................... 20 gAdd 320mOSM Phosphate buffer to make the 1 L solution

This fixative is used primarily for immunohistochemistry and electron microscopy, and is suited for fixing polypeptide antigens. Fixation takes about 4 to 12 hours at 4°C. After fixation, wash specimen with 70% ethanol until the yellow color disappears.

Data

Product Name Grade Package Size Wako Cat. No.Bouin Solution for Pathology Research 1 L 023-17361

Carnoy Solution for Tissue Fixation 1 L 034-17711

4% Paraformaldehyde Phosphate Buffer Solution for Tissue Fixation100 mL 161-20141500 mL 163-20145

PLP Solution Set for Tissue Fixation for 250 mL 290-63201

Zamboni Solution for Pathology Research 1 L 263-01991

A-2. for Electron Microscopy

Fixatives for Electron Microscopy

Fixatives Working Concentration Advantage Disadvantage Applications

Formalin Neutral Buffer 4%

Can effectively fix soluble proteins (e.g. functional proteins such as hormones and enzymes) and polypeptides

- Soluble protein, polypeptide

Glutaraldehyde 1.5~4%

Enables negative imaging of intracellular structure, glycogen granules; Enables positive imaging of nuclear and intracel-lular matrices

More time required for washing and dehydrating; weak penetra-tion of embedding materials other than methacrylic resin; inadequate fixation of lipids; no fixation of proteins at low concentrations

Polysaccharides

Osmic Acid 1~2%

Reduces washing and dehydra-tion time; less deformation during fixing; good embedding agent penetration enables use of various types of resin; ideal for fixing phospholipids

Weak penetration; causes loss saturated fatty acids and 50% of all proteins

Intracellular microstructures; unsaturated fatty acids; DNA granules

Paraformaldehyde 4%Can be used for fixing glycopro-tein and preserving antigenicity; penetrates tissue rapidly

Weak fixation Protein antigens

PLP (Periodate-lysine-PFA) 2%

Suitable for fixing glycoprotein and sugar; Can keep antigenicity

- Glycoprotein antigens

Bouin Solution Fixation: mouse testicle

HE Staining CD4/MT310 Immunostaining HE Staining Insulin immunostaining

Zamboni Solution Fixation: rat pancreas

PLP fixation: mucous membrane of the colon


AFi

xativ

es

A-2. for Electron MicroscopyFixatives for Electron Microscopy

B. Tissue Dehydrating Solution

I n the course of preparing pathology specimens, tissues are thinly sliced after paraffinization and embedding block assembly. In the same manner that water and oil do not mix, paraffin, an oil derivative, will not penetrate wet tissue

samples. Therefore, it is necessary to dehydrate the specimen with alcohol and then replace the alcohol with an interme-diate. Consequently, alcohol is an indispensable solvent for preparing pathology specimens. However, after amendment of the Liquor Tax Act in 1994 resulted in higher ethanol prices researchers began looking for a cheaper alternative with the same properties. Our tissue dehydrating solutions are made of denatured alcohol with reduced water content and provide the same performance as ethanol. Moreover, these products are affordable because they do not fall within the purview of the Liquor Tax Act. Product specifications are listed below.

for Electron Microscopy Tissue Dehydration Solution 99 Tissue Dehydration Solution 100

Component

Ethanol: 99+%Acetone: 0.7%Dehydration Agent: Zeolite 3Å type abt. 3g packed in a bagDenaturant: containing Bitrex™: 10 ppm

Ethanol: 99.8+%Dehydration Agent: Zeolite 3Å type abt. 3g packed in a bagDenaturant: containing Bitrex™: 10 ppm

Water ≤ 0.2% ≤ 0.2%Feature the same purity and performance of conventional ethanol

Product Name Grade Package Size Wako Cat. No.Tissue Dehydration Solution 99 for Electron Microscopy 500 mL 205-17445

Tissue Dehydration Solution 100 for Electron Microscopy 500 mL 208-17435

Fixatives Working Concentration Advantage Disadvantage Applications

Potassium permanganate 0.6~3%

As a fixative, exhibits medium penetration of the embedding material

Cases much deformation and partial loss of mucopolysac-charides, proteins, saturated fatty acids and phospholipids; does not fix ribosomes

Intracellular microstructures; glycogen granules; plant specimens

Tannic Acid, Aldehyde 1%, 2%

Can effectively fix soluble proteins (e.g. functional proteins such as hormones and enzymes) and polypeptides when mixed with an aldehydes fixative

- Soluble protein, polypeptide

Product Name Grade Package Size Wako Cat. No.10% Glutaraldehyde Solution for Electron Microscopy 2 mL × 5 071-02031

20% Glutaraldehyde Solution for Electron Microscopy25 mL 072-02262

500 mL 076-02265

70% Glutaraldehyde Solution for Electron Microscopy 2 mL × 5 071-01931

Osmium (VIII) Oxide for Electron Microscopy 1 g 154-01014

2% Osmium (VIII) Oxide Solution for Electron Microscopy 5 mL × 5 154-01151

4% Osmium (VIII) Oxide Solution for Electron Microscopy 5 mL × 5 157-01141

Paraformaldehyde for Tissue Fixation100 g 160-16061500 g 162-16065

2,4,6-Trimethylpyridine [γ-Collidine] for Electron Microscopy 25 mL 035-08093

Tissu

e Deh

ydrat

ing

Solut

ionB


Decalcifying Agent

C

C. Decalcifying Agent

U sing microtome blades to prepare thin sections of hard tissue such as bone and calcified foci is a formidable challenge. Decalcifying the specimen in advance makes the process easier. Thin slicing can be done more efficiently

if the calcium carbonate in the tissue is dissolved with acid or some other solvent and converted into a softer salt. A proper decalcifier needs to possess the following properties. l Must decalcify rapidly l Must not affect tissue stainingl Must be easy to use l Must not cause tissue expansion, contraction, dissolution or other types of damage

Basic ProtocolResection ➡ Dehydrate/Delipidate ➡ Hydrate ➡ Wash ➡ Decalcify ➡ Neutralize ➡ Dehydrate ➡ Remove alcohol ➡ Paraffinize ➡ Slice ➡ Deparaffinize ➡ Stain

l Thickness of tissue: Preferably more than 5 mm.l Decalcifier concentration: 100 mL or more for 1 gl Decalcifying temperature:

As a rule, perform at room temperature. Although higher temperatures will result in faster decalcification, more tissue damage will also occur. Often used at 30°C, Decalcifier B (neutral EDTA) has been reported to preserve the original tissue structure and provide regular staining performance at 56°C.

l Neutralization: Neutralization with 5% sodium sulfate will be necessary when employing Kalkitox™, Decalcifying Solution A (Plank-Rychlo protocol), hydrochloric acid or nitric acid. In general, neutralization will take the same length of time as decalcification; however, schedule at least half a day if you want faster results. After neutralization, thoroughly wash away the sodium sulfate solution with water.

l Other tips: Agitating the specimens two or three times a day will speed up the decalcification process.

Kalkitox™ This product is a new type of decalcifier that combines the advantages of the Plank-Rychlo and chelating agent methods.

InstructionsDecalcification takes about one night at room temperature, although this will depend to a certain extent on the size of the specimen. With Kalkitox, we recommend low-temperature decalcification (refrigeration at 4°C for one or two nights), followed by neutralization in sodium sulfate solution for several hours to one night, and then thorough washing with water. Afterwards, perform dehydration, solution replacement with an intermediate and then paraffinization.

Advantagesl Enables simultaneous neutralization and decalcification l Enables low-temperature decalcification at 4°C l Enables immunostaining

CompositionMain ingredients are hydrochloric acid and EDTA.

Data

Fig. 1: Bone metastasis of colorectal adenocarcinoma (×10) Left: HE staining; Right: Alcian Blue, PAS staining

Fig. 2: Femoral osteosarcoma Top left: Masson trichrome stain Bottom left: Silver impregnation stain

Right: Immuno-enzymatic method (Top right: Osteocalcin; Bottom right: Osteonectin)

Product Name Grade Package Size Wako Cat. No.Kalkitox™ for Pathology Research 1 L 112-00651


Deca

lcify

ing

Agen

tC

Decalcifying Solution A, B Decalcification Decalcifying Solution Composition Features

Rapid Decalcification Decalcifying Solution A (Plank-Rychlo protocol)

l Aluminum Chloride ............ 7.0 gl Hydrochloric Acid .............. 8.5 mLl Formic Acid .......................... 5.0 mLPrepare the 100 mL solution with distilled water

l �Processing time is about 3x the length for formic or hydrochloric acid

l Poor maintenance of antigenicityl Requires neutralization with 5% sodium sulfate

Neutral Decalcification Decalcifying Solution B (EDTA protocol)

Main component: 0.5 mol/L EDTA solution (pH 7.0 ~ 7.5)

l Less damage to tissuel Good preservation of structure and stainingl Ideal for immunohistostainingl Decalcification requires a longer length of time

DataDecalcifying Solution A Decalcifying Solution B

He Staining

Complete decalcification after 12 hoursbone thickness of 10 mm

Fast decalcification

Complete decalcification after 5 daysbone thickness of 3 mm

Slow decalcification

Immunohistostaining

Complete decalcification after 12 hoursbone thickness of 3 mm

Loss of carcinoembryonic antigen sites

Complete decalcification after 5 daysbone thickness of 3 mm

Carcinoembryonic antigen sites are kept.

Product Name Grade Package Size Wako Cat. No.Decalcifying Solution A (Plank-Rychlo protocol) for Pathology Research 1 L 047-21911

Decalcifying Solution B (EDTA protocol) for Pathology Research 1 L 047-21911

Morse Solution Sections of bone tissues, calcification foci, and calcium deposited lesions cannot be prepared as they are. Therefore, they need decalcification. In this procedure, tissue calcium carbonate is degraded to soft calcium salts with an acid allowing easy sectioning. This product decalcifies 8-time more quickly than neutral EDTA solution and the results are said to be comparable to EDTA in immunostaining and in situ hybridization.

Product Name Grade Package Size Wako Cat. No.Morse Solution for Pathology Research 1 L 135-17071

<Related Products>Product Name Grade Package Size Wako Cat. No.

5% Formic Acid (for Decalcification) for Pathology Research 500 mL 062-04025

10% Hydrochloric Acid (for Decalcification) for Pathology Research 500 mL 089-07675

8% Nitric Acid (for Decalcification) for Pathology Research 500 mL 146-07075

5% Sodium Sulfate Solution (for Decalcification) for Pathology Research 500 mL 196-11985

5% Trichloroacetic Acid Solution (for Decalcification) for Pathology Research 500 mL 205-14905

Rat nasal cavity HE Staining

Compositionl 22.5% Formic Acidl 10% Sodium Citrate


Intermediates

(Replacing agents)D

D. Intermediates (Replacing agents)

A lthough you may have replaced the water in the dehydrated tissue with alcohol, you will need to replace this alcohol with a solution compatible with paraffin. This is because alcohol and paraffin do not mix well. Currently popular

intermediates include alcohol and paraffin-compatible xylene. However, xylene is highly volatile, foul-smelling and hazardous to health. These adverse properties have made use of paraffin in the workplace a controversial issue. Lemosol® and Lemosol® A are alternatives to xylene that are virtually non-toxic and do not emit offensive odors. Lemosol® A is particularly unique because it contains phytoncide, the substance responsible for the pleasant odor of forests.

D-1. Lemosol®, Lemosol® A (Ace)

Decalcification Lemosol® Lemosol® A (Ace)Main component D(+)-Limonene PineneOrigin orange peel bark of eucalyptus and pineSmell Citrus Eucalyptus and pine resinVolatility Less volatile compared to xylene Superior volatilitySpecific Gravity (20°C) 0.838 ~ 0.844 g/mL 0.873 ~ 0.879 g/mLFlashing Point 61°C 36.5°C

Toxicity (LD50 rat p.o.)

≥ 5 g/kgDoes not irritate the eyes, nose, throat or skin.Limonene has been approved by the U.S. Food and Drug Agency as a food additive and flavoring agent.

≥ 5 g/kgDoes not irritate the eyes, nose, throat or skin.Pinene has been approved by the U.S. Food and Drug Agency as a food additive and flavoring agent.

Boiling Point 177°C (d-Limonene) 154~166°CRefractive Index (20°C) 1.473 1.475

n Lemosol® Lemosol® is a safe substitute for xylene and chloroform. Its pleasant lemon fragrance comes from one of its main ingredients, D-(+)-Limonene, which is a substance derived from orange peel oil.

Featuresl Safe for human usel Can be used as an intermediate in place of xylene and chloroform, as well as a deparaffinizing agent and a clearing agentl �In cases where use of xylene will lead to tissue hardening and contraction, Lemosol® can keep organs soft for prepara-

tion of paraffin-embedded sectionsl Unlike xylene, Lemosol® is decomposed by microorganisms, resulting in easier disposall Safely compatible with conventional staining methods and immunohistostaining protocolsl Contains refined limonene which emits a mild citrus fragrance

Examples of stained sections prepared with Lemosol®

Human stomach (biopsy)

HE Staining

Human lung (CEA)

ABC Staining

n�Lemosol® A (Ace) Lemosol® A is terpene-based solvent derived from bark of eucalyptus and pine.Lemosol® A can be used in place of xylene as an intermediate and clearing agent for the preparation of pathology specimens. An advanced version of Lemosol®, this product and does not emit offensive odors and exhibits good volatility.

Featuresl Superior volatility l Does not irritate the eyes, nose, throat or skin.l Applicable to HE staining, special staining and immunohistostainingl Can be solidified and disposed after use

Main component: Pinene compound

Main component: D-(+)-Limonene


Inte

rmed

iate

s (Re

placin

g age

nts)

D

D-1. Lemosol®, Lemosol® A (Ace)Lemosol® A (Ace)

Example of a protocol employing an intermediate

70% Alcohol for 1 hr.70% Alcohol for 1 hr.70% Alcohol for 1 hr.70% Alcohol for 1 hr.

100% Alcohol

I for 1 hr.II for 1 hr.III for 1.5 hr.IV for 1.5 hr.

IntermediateI for 1 hr.II for 1 hr.

ParaffinI for 1 hr.II for 1 hr.

Paraffin [Melting container at 62°C]

III for 10 min.IV for 10 min.

Paraffin [vacuum] V for 10 min.

Conditions for deparaffinization and alcohol processing

IntermediateI for 2 ~ 3 min.II for 3 ~ 5 min.III for 3 ~ 5 min.

100% AlcoholIIIIII

Wash

Conditions for clearing and mounting

100% AlcoholIIIIII

IntermediateI for 1 hr.II for 1 hr.III for 1 hr.

Clear with xyleneMount

Disposal method for Lemosol® and Lemosol® A① We can provide a coagulant for safe disposal.② Flushing solution down a drain that directly leads to public waters will result in a rise of BOD and TOC. Therefore, if

you are planning to dispose of solution in its liquid form, treat beforehand with activated sludge or some other appropriate method.


Lemosol® for Pathology Research1 L 122-039913 L 128-03993

Lemosol® A (Ace) for Pathology Research1 L 120-044113 L 126-04413

n�Lemosol® Coagulant This product is a coagulant for the safe disposal of Lemosol® and Lemosol® A (Ace), which are used as intermediates or clearing agents for the preparation of pathology specimens.

Main ComponentHydrogenated castor oil fatty acid

Basic Protocol① Add an amount of Lemosol® coagulant that is approximately 5% the volume of the Lemosol® or Lemosol® A to be

disposed.② Place the coagulant and solution mixture on a hot plate stirrer or in a hot water bath. Heat to about 60°C and agitate

in order to melt the contents. (Be careful not to cause a fire.)③ Cool the mixture until solid.

<Note>l �Both Lemosol® and Lemosol® A are highly flammable. Do not expose to open flames. Moreover, both products should not be handled

near any type of flame. l After coagulation, handle the generated waste as a Class II Hazardous Material No. 1 flammable solid. l �Waste solution containing xylene (regulated as a deleterious material for non-medical use and as a hazardous substance under the Fire

Service Act) or chloroform (regulated as a deleterious material for medical use) cannot be disposed in the manner described above.

<Specific measures>l If you are planning to store the coagulated solution indoors for a period of time:

Place the coagulated solution in a vinyl bag so the contents do not leak. Seal the bag tightly at the opening and store in a location away from any open flames.(The contents may vaporize, especially during summer. Therefore, if the bag is not properly sealed, leaking vapors may combust on contact with an open flame.)

l If you are planning to dispose the coagulated solution immediately (In case of Japan):If the amount to be disposed at a single time is less than 20 kg, we recommend you use your regular facilities to process the waste. No special permission from the local authorities is necessary. If the amount to be disposed at a single time is from 20 kg to less than 100 kg, you need to obtain special permission from the local authorities.

Product Name Grade Package Size Wako Cat. No.Lemosol® Coagulant for Solidification of Used Lemosol 500 g 129-04545


Embedding

Medium

E

E. Embedding Medium

Pathoprep® Series Paraffin for embedding of pathologic tissue. Pellet type.Purified paraffin with uniform carbon distribution, to which a safe and stable high-molecular polymer has been added to increase tissue permeability. This line of products is superior in terms of sliceability, extensibility and penetration.

Pathoprep® 546 Pathoprep® 568 Pathoprep® 580

M.P.: 54 ~ 56°C; DMSO-free M.P.: 56 ~ 58°C; DMSO-free M.P.: 58 ~ 60°C; DMSO-free

Product Name Grade Package Size Wako Cat. No.Pathoprep® 546 for Pathological Tissue Embedding 2 kg × 3 167-20501

Pathoprep® 568 for Pathological Tissue Embedding 500 g × 12 162-18961

Pathoprep® 580 for Pathological Tissue Embedding 2 kg × 3 165-19551

Other Embedding MediumProduct Name Grade Package Size Wako Cat. No.

Collodion (5%) Wako 1st Grade 500 mL 032-03885

Collodion (10%) Wako 1st Grade 500 mL 031-16425

Gelatin Wako 1st Grade 500 g 077-03155

Embedding Medium for Electron MicroscopyProduct Name Grade Package Size Wako Cat. No.

Ethylene Glycol Diglycidyl Ether Electron Microscopy200 g 056-03841500 g 058-03845

Epon 815 Electron Microscopy200 g 056-02981500 g 058-02985



<Related Products>hardening Agents


Dodecenylsuccinic Anhydride [DDSA] Electron Microscopy200 g 040-16251500 g 042-16255

Methyl-5-norbornene-2,3-dicarboxylic Anhydride [MNA] Electron Microscopy

200 g 134-05951500 g 136-05955

Nonenylsuccinic Anhydride [NSA] Electron Microscopy100 g 141-04541500 g 143-04545

AcceleratorProduct Name Grade Package Size Wako Cat. No.

2,4,6-Tris (dimethylaminomethyl) phenol [DMP-30] Electron Microscopy 25 mL 204-06263

Gelatin, CapsulesProduct Name Grade Package Size Wako Cat. No.

Gelatin, Capsules No.0 Electron Microscopy 100 ea. 074-01801

Gelatin, Capsules No.00 Electron Microscopy 100 ea. 071-01791


Imm

unop

enF

F. Immunopen

Immunopen is a felt-tipped pen type reagent and used as an adjunct to immunostaining. It is designed to minimize waste of antiserum in immunostaining techniques on sections mounted on slide glasses and to induce immunoreactions efficiently.

An example for use <Section on a slide glass plate>1. Deparaffinizing2. Wash in distilled deionized water.3. Immunopen Treatment: Draw a circle around the section on the slide glass. It becomes a barrier to protect unnecessary spreading of solutions for staining.

Direction for use1. Used as a tool for immunostaining2. Used in ABC staining and fluorescent antibody stanining

Precautions1. Immunopen is insoluble in water. Before use, thoroughly wipe up water around sections with filter papers on a slide glass.2. Immunopen is soluble in oil. When tissue sections are fixed in acetone, use Immunopen

after fixation and solvent removal.

Product Name Package Size Wako Cat. No.Immunopen 5 pens 295-20553


StainsG

G. Stains

Purpose of stain Stain Method Stain Result Wako’s Products Page

General stains (G-1)

Hematoxylin-eosin (HE) stain

Nuclei, cartilage, bacteria, non-decalcified areas : Indigo blueCytoplasm, connective tissue, muscle tissue, red blood cells : pink to reddish pink

Mayer's Hematoxylin SolutionMayer's Hematoxylin Solution (×2)Carrazzi's Hematoxylin Solution (×2)Eosin Alcohol Solution, acid extract1% Eosin Y Solution0.1% Eosin Y Ethanol Solution

14

Polysaccharides (G-2)

Glycogen PAS stain

PAS-positive substances : Reddish purplePAS-positive substances: glycogen, proteins (e.g.

mucin, basement membrane, fibrin compounds, some hormones, lipofuscin, fungi, Entamoeba histolytica)

Schiff's ReagentCold Schiff's Reagent 16

Acidic slime polysaccharides

Alcian blue stain Acid mucopolysaccharide : blueNuclei : red Alcian Blue Solution, pH2.5 17

Toluidine blue stain

Acid mucopolysaccharide, mast cell granule : red to reddish purple (metachromasy)Nuclei, other tissue components : blue

0.05%Toluidine Blue Solution (pH 2.5)0.05%Toluidine Blue Solution (pH 4.1)0.05%Toluidine Blue Solution (pH 7.0)

18

Colloidal iron stain Acid slime polysaccharide : blueNuclei : red Colloidal Iron Stock Solution 19

Connective tissues (G-3)

Collagen fibers

Elastica-van Gieson stain

Collagenous fiber, reticular fiber, basement membrane : redMuscle fiber, cytoplasm : yellowNuclei : blackish brown

Weigert's Iron Hematoxylin Staining SetVan Gieson Solution PVan Gieson Solution F

19

Masson’s trichrome stain

Collagenous fiber, reticular fiber, renal glomerular basement membrane : blueNuclei : purplish black to purplish redCytoplasm : pale red to purplish red

Aniline Blue Solution0.8% Orange G SolutionPonceau Xylidine. Acid Fuchsin. Azophloxine Solution

20

Azan stain

Collagenous fiber, reticular fiber, renal glomerular basement membrane : bright blueNuclei : dark redFibrin : red

Aniline Blue · Orange G SolutionAzocarmine G Solution 21

Elastic fibers Victoria blue stain

Elastomeric fiber : HBs antigenCartilaginous matrix : blueNuclei : blueBackground : pale pink

Victoria Blue Solution 22

Fibrin, glial tissues (G-4)

Phosphotungstic Acid Hematoxylin (PTAH) stain

Glial fiber, fibrin, striated muscle striations, nucleus, smooth muscle fiber : bluish indigoConnective tissue, nerve cell :

brownish red

Phosphotungstic Acid Hematoxylin Solution 23

Intracellular inorganic substances (G-5)

Iron (hemosiderin) Berlin blue stain Hemosiderin : blue

Nucleus : red Berlin Blue Staining Set 23

Cytodiagnosis (G-6) Papanicolaou stain Nucleus : dark purple

Papanicolaou-Gill's Hematoxylin (Gill-5) Stain SolutionPapanicolaou, Hematoxylin Stain SolutionPapanicolaou EA100 Stain SolutionPapanicolaou OG100 Stain Solution

24

Hematocyte(G-7)

Giemsa StainWright StainWright-Giemsa StainMay-Grünwald Stain

Nuclei : reddish purple

Giemsa Stain SolutionWright Stain SolutionMay-Grünwald Stain SolutionMay-Grünwald's Eosin Methylene Blue Solution

25

Hard tissue (G-8)

Stains for osteoblast, osteoclast

TRAP/ALP stain Osteoblast : blue to brownish blueOsteoclast : reddish purple TRAP/ALP Stain Kit 26

Osteoid stains Villanueva Bone Stain Osteoid : reddish purple -


Stai

nsG

G-1. General Staining

Hematoxylin-Eosin (HE) Stain Hematoxylin-eosin (HE) stain is a method to stain the nucleus dark blue with hematoxylin and the cytoplasm dark and pale red with eosin, and is used for overview staining to know the entire cell and tissue structures.

Principle of StainsIt is considered that hematin generated by oxidizing hematoxylin forms a complex with the metallic portion of the mordant, is charged positively, and is then bound to the negatively charged phosphate group of the nucleus to produce staining. (It is also considered that the cytoplasm and connective tissue are charged positively and are bound to the negatively charged eosin: afterstaining with eosin)

n�Mayer’s Hematoxylin Solution This product is the most widely used Mayer’s hematoxylin solution. Double-strength Mayer’s hematoxylin ((×2) product) contains twice the amount of hematoxylin, enabling faster staining.

CompositionHematoxylin ....................................... 1.0 g (In case of the (×2) product: 2.0 g)Potassium Alum ................................50 gSodium Iodate .................................. 0.2 g (In case of the (×2) product: 0.4 g)Chloral Hydrate ..................................50 gCitric Acid ............................................ 1.0 gDistilled Water ..............................1,000 mL pH 2.0 ~ 3.0 (25°C)

<Note>If crystals of aluminum potassium sulfate 12-hydrate precipitate during preservation, use a supernatant solution.

Product Name Grade Package Size Wako Cat. No.Mayer’s Hematoxylin Solution for Pathology Research 500 mL 131-09665

Mayer’s Hematoxylin Solution (52) for Pathology Research 500 mL 134-13065

Renal biopsy (×200)

Hematoxylin and Eoxin (HE) Staining Protocol

Paraffin Section

Deparaffinize, Wash for 5 min.

Hematoxylin Stain for 3 ~ 5 min.

Stain Enhancement (Warm water) for 5 min.

Eosin Solution for 5 min.

Differentiationk

95% Alcohol

I for 3 ~ 5 min.

II for 3 min.

III for 3 min.

100% Alcohol

I for 3 ~ 5 min.

II for 3 ~ 5 min.

III for 3 min.

Clear and Mount

k�: A procedure involving the use of alcohol to decolorize stained areas other than the target components.

Hematoxylin Hematin

Nucleus


StainsG

Hematoxylin-Eosin (HE) Stain G-1. General Staining

CompositionAcid-extracted Eosin ............. 100 mL95% Alcohol ................................ 800 mLAcetic Acid .......................................... 8 mL

l 1% Eosin Y SolutionEosin Y ..............................................5.0 gDistilled Water ............................. 500 mLAcetic Acid ...........................A few drops

l 0.1% Eosin Y Solution10% Eosin Y Solution ....................5 mL95% Alcohol ................................. 495 mL

l 0.5% Eosin Y Solution10% Eosin Y Solution .................25 mL95% Alcohol ................................. 475 mL

Examples of staining

Product Name Grade Package Size Wako Cat. No.Eosin Alcohol Solution, acid extract for Pathology Research 1 L 050-06041

n�Eosin Alcohol Solution, Acid Extract This product is alcohol solution of acid-extracted eosin for good contrast staining. It can be used immediately allowing clear staining of fine cellular structures.

n�Eosin Y Solutions

The above mentioned three type products show excellent staining performance. The aqueous solution type in particular is also applicable to cryosection. In general, higher concentrations are recommended for thinner sections.

Product Name Grade Package Size Wako Cat. No.1% Eosin Y Solution for Pathology Research 500 mL 051-06515

0.1% Eosin Y Ethanol Solution for Pathology Research 500 mL 054-06505

0.5% Eosin Y Ethanol Solution for Pathology Research 500 mL 051-06495

n�Carrazzi’s Hematoxylin Solution Carazzi’s Hematoxylin is used primarily as a cytodiagnostic tool because, with the proper differentiation protocol, it can stain intranuclear structures with remarkable clarity.

CompositionHematoxylin ....................................... 1.0 g (In case of the (×2) product: 2.0 g)Potassium Alum ................................50 gSodium Iodate .................................. 0.2 g (In case of the (×2) product: 0.4 g)Glycerin ................................................. 200 mLDistilled Water .................................. 800 mL

Product Name Grade Package Size Wako Cat. No.Carrazzi's Hematoxylin Solution for Pathology Research 500 mL 032-14635

Carrazzi's Hematoxylin Solution (52) for Pathology Research 500 mL 039-17705

Composition

Using Acid extracted Eosin Using conventional Eosin

Human Kidney


Stai

nsG


Schiff’s Reagentfor Pathology Research 100 mL 191-08441for Pathology Research 500 mL 193-08445

G-2. Polysaccharide Staining

PAS (Periodic Acid Schiff) Stain n�Schiff’s Reagent A Schiff reagent is a stain reagent used frequently in the PAS (Periodic Acid Schiff) reaction and Feulgen’s reaction in the fields of histopathology, clinical pathology and hematology. As it reacts with aliphatic aldehydes sensitively and develops red to purple colors, it is applied extensively to intracellular staining, staining of polysaccharides such as neutral mucopoly-saccharides, glycoproteins, etc. and staining of lipids constituting lipoproteins. There are two types of Schiff reagents according to preparation protocols. One is hot Schiff reagent which is prepared by boiling. The other is cold Schiff reagent which is prepared at room temperature. Although the preparation protocols differ, there is virtually no change in staining performance.

Advantagesl Highly sensitive to aliphatic aldehydesl Good reproducibilityl Excellent preservation stability

Reaction Formula

Paraffin Section

Deparaffinization/Wash

0.5% Periodic Acid for 5 ~ 10 min.

Wash with running water for 3 min.

Rinse with Distilled Water 3 ~ 5 times

Schiff’s Reagent for 10 ~ 15 min.

Sulfurous acid solutionk

I for 3 min.

II for 3 min.

III for 3 min.

Wash with running water for 3 ~ 5 min.

Nucleus stain with Hematoxylin for 3 min.

Stain Enhancement with running water for 5 ~ 10 min.

Clear/Mount

CompositionFuchsin Basic ....................................................... 10 gSodium metabisulfite ...................................... 19 g0.5 mol/L Hydrochloric acid ...............1,000 mL

k�: Sulfurous acid solutionl 10% sodium sulfite (or 10% sodium bisulfate); 6 mLl 1 M hydrochloric acid: 5 mLl Distilled water: 100 mL

n�Cold Schiff’s Reagent In comparison to hot Schiff reagent, cold Schiff reagent is prepared at room temperature and is better for staining sections with a thickness of 2 μm or less.

Staining resultsGlycogen stains deep purplish red. Mucin stains red to purple. Fibrin stains pink. Reticular tissue and basement membrane stain purplish red. PAS-positive microorganisms include fungi (yeast, blastomyces), bacteria (e.g. anthrax, gut flora) and Entamoeba histolytica.

CompositionFuchsin Basic ................................................................ 10 gSodium metabisulfite ............................................... 19 g0.15 mol/L Hydrochloric acid .....................1,000 mL

Product Name Grade Package Size Wako Cat. No.Cold Schiff’s Reagent for Pathology Research 500 mL 039-14645

Aldehyde

Schiff’s Reagent Quinoid chromophore (red ~ purplish blue)

Polysaccharide

Human Kidney

Human Kidney


StainsG


Tissue Section

Deparaffinize/wash

3% Acetic Acid for 3 min.

Alcian Blue Staining Solution for 30 min.


Nucleus stain with Hematoxylin

Wash

Dehydrate

Clear/Mount

Deparaffinize/wash

3% Acetic Acid for 3 min.

Alcian Blue Staining Solution for 10 ~ 30 min.

Wash with running waterk1

Distilled Water

0.5% Periodic Acid for 5 ~ 10 min.

Wash with running water

Cold Schiff’s Reagent for 15 ~ 30 min.

Sulfurous Acid Solutionk2

I for 3 min.

II for 3 min.

III for 3 min.


Nucleus stain with Hematoxylin

Wash/Stain enhancing

Dehydrate/Clear/Mount

Stain Result with Alcian Blue Solution (pH 2.5)

Double-Staining with Alcian Blue Solution (pH 2.5) and Cold Schiff’s Reagent

Staining Resultl Acidic mucopolysaccharides: bluel Neutral mucusl Sialomucin, sulfomucin: Reddish purple ~ bluel Nucleus: bluish purple ~ purplish indigo

<Note> If you prepare the pH of Alcian blue to 1.0, only sulfomucin and acid mucin will stain reddish purple to blue, and neutral mucin will stain bright red.

k1:�Thoroughly wash with distilled water after washing with running water.k2:�Refer to the Schiff’s Reagent page of the PAS-staining protocol (See the page #15).

Product Name Grade Package Size Wako Cat. No.Alcian Blue Solution, pH 2.5 for Pathology Research 500 mL 015-13805

Alcian Blue Stains Alcian blue stains mucopolysaccharides by binding specifically to their sulfate and carboxyl groups. At pH 2.5, both the sulfate and carboxyl groups are stained, whereas at pH lower than 1.0, only the sulfate group is known to bind with alcian blue.The alcian blue solution is used for double-staining with PAS, which makes it possible to stain neutral and acid mucopoly-saccharides in different colors.Each cell and tissue produces specific acid mucopolysaccharides, and by observing the tissue-specific acid mucopolysac-chrides, it is possible to distinguish between particular characteristics of each tumor tissue.

Human Large Intestine

Human Stomach


Stai

nsG


Toluidine Blue Staining Acidic mucopolysaccharides are present in the connective tissue (hyaluronic acid and chondroitin sulfate B), cartilage tissue (mucoitin sulfate, chondroitin sulfate A,C), mast cells (heparin), and epithelial mucus, etc.The staining method is based on the fact that these compounds stain metachromatically with the basic tar dye toluidine blue.

Deparaffinize

100% Alcohol I, II, III

95% Alcohol

70% Alcohol

Wash with Distilled water after washing with running water

0.005% Toluidine Blue Solution (for 30 minutes)

100% Alcohol I, II, III, IV

Clear/Mount

Composition0.05% Toluidine Blue Solution: pH 2.5, 4.1 and 7.0Reagent 1: 0.1 mol/L Citric Acid SolutionReagent 2: 0.2 mol/L Disodium Hydrogen Phosphate Solution

l �Mix Reagent 1 and Reagent 2 in the ratios described below and prepare buffers. Add toluidine blue at a proportion of 0.05% to each solution and dissolve.

pH 2.5 Solution: <Reagent 1> : <Reagent 2> = 19.0: 1.0 (mL)pH 4.1 Solution: <Reagent 1> : <Reagent 2> = 12.0: 8.0 (mL) pH 7.0 Solution: <Reagent 1> : <Reagent 2> = 3.5: 16.5 (mL)

Mucus

pH

Acidic Mucopolysaccharide

Hyaluronic Acid Mucoitin SulfateChondroitin Sulfate

2.5 - +4.1 + +7.0 + +

Staining Protocol

Staining ResultAcidic mucopolysaccharides, mast cell granule: reddish purpleNuclei: blueSulfur-containing poysaccharides such as chon-droitin sulfate show positive metachromasy (reddish purple) at all pH. On the other hand, hyaluronic acid shows a negative metachromasy (blue) at pH 2.5.

Product Name Grade Package Size Wako Cat. No.0.05% Toluidine Blue Solution (pH2.5) for Pathology Research 500 mL 202-14535

0.05% Toluidine Blue Solution (pH4.1) for Pathology Research 500 mL 209-14545

0.05% Toluidine Blue Solution (pH7.0) for Pathology Research 500 mL 206-14555

[Fig. 1, 2] Metachromasy of the umbilical cord(0.05% Toluidine Blue, pH 4.1)Metachromasy of hyaluronic acid molecules that have not conjugated with sulfate groups and of acid mucopolysaccharides

[Fig. 3, 4] Metachromasy of the tracheal cartilage(0.05% Toluidine Blue, pH 2.5, pH 4.1)Enhanced metachromasy of sulfomucopolysaccharides

[Fig. 5, 6] Metachromasy of heparin in mast cells(0.05% Toluidine Blue, pH 4.1)

Data was provided by Teiji Takezaki & Masahiko Fujiwara, Pathological and Microbiology Lab, Kotobiken Medical Laboratories

[Fig.1] Toluidine Blue, pH 4.1 (×4)







StainsG

Colloidal Iron Staining The colloidal iron staining is a method in which colloidal trivalent iron (Fe2O3) is adhered to acid mucopolysaccharides and detected by Prussian blue reaction.

n�Weigert’s Iron Hematoxylin Staining Set Kit Components

Weigert’s Iron Hematoxylin Solution I .......... 500 mL × 1 bottle (1% Hematoxylin-96% Ethanol)Weigert’s Iron Hematoxylin Solution II ........ 500 mL × 1 bottle (2% Iron (III) Chloride, Hexahydrate- abt. 0.25% Hydrochloric Acid)

Preparation of ReagentStaining Solution (Prepare just before use)Mix Solution I and II at a ratio of 1:1.

Product Name Grade Package Size Wako Cat. No.Colloidal Iron Stock Solution for Pathology Research 100 mL 037-11601

Deparaffinize

Distilled Water 2 or 3 times

Colloidal Iron Solutionk1 2 hr.

for 10 min. × 3 times

Distilled Water for 5 min. × 3 times

Potassium Ferrocyanide/Hydrochloric Acid Solution 10~20 min.

Wash with running water 1 time

Kernechtrot (Nuclear fast red) 2 times

Wash with running water Gently


Staining Resultl Elastic fibers, HBs antigens, cartilage matrix: Bluel Nuclei: Redl Background: Light pink

k1: Colloidal Iron Solution Distilled Water ..............................................18 mL Glacial Acetic Acid .....................................12 mL Colloidal Iron Stock Solution .................10 mL

Staining Resultl Elastic fibers: Blackish purplel Collagen fiber: Redl Muscle fiber, Erythrocyte, Cytoplasm: Yellowl Nucleus: Blackish purple

G-3. Connective Tissue Staining

Elastic Van Gieson’s Stain Collagen fibers undergo the most rapid growth when damaged connective tissue is being repaired. The standard Van Gieson’s stain is a widely used method of visualizing such collagen fibers. However, most investigators want to stain more than one target. In such cases, the advanced Elastic Van Gieson’s stain is more advantageous and popular because it allows simultaneous histology of elastic, collagen and muscle fibers.

Product Name Grade Package Size Wako Cat. No.Weigert's Iron Hematoxylin Staining Set for Pathology Research 1 set 298-21741

Deparaffinize

Weigert's Resorcin-Fuchsin solution 30 min. ~ 12 hr.

100% Alcohol

I for 3 min.

II for 3 min.

III for 3 min.


Weigert’s Iron Hematoxylin Solution for 5 ~ 10 min.


Van Gieson Solution for 2 ~ 3 min.

70% Alcohol 3 times

90~100% Alcohol 2 times each

Clear/Mount

Human Kidney


Stai

nsG

Elastic Van Gieson’s Stain G-3. Connective Tissue Staining

n�Van Gieson Solution P (Picric Acid Solution) Composition

A solution with picric acid dissolved in distilled water to the saturation point.

n�Van Gieson Solution F (1% Acid Fuchsin Solution) Composition

Acid Fuchsin ......................................5 gDistilled Water ............................. 500 mL

➡ Prepare Van Gieson’s solution just before use. (Van Gieson Solution P : Van Gieson Solution F = 100 mL : 15 mL)

Masson’s Ttrichrome Stain Masson’s trichrome stain is a method of staining to distinguish collagen fibers. Nucleus is stained black with iron hema-toxylin, cytoplasm is stained red with Xylidine Ponceau acid fuchsin azofloxin, and collagen fiber is stained blue with aniline blue. It is called “trichrome stain”.

Product Name Grade Package Size Wako Cat. No.Van Gieson Solution P for Pathology Research 500 mL 224-01405

Van Gieson Solution F for Pathology Research 500 mL 221-01415

Deparaffinize

Xylene IXylene IIXylene III

5 min. each

100% Alcohol I100% Alcohol II100% Alcohol III70% Alcohol

2~3 min. each

Dealcoholize/Wash Running water → Distilled water hydrate

1st Mordant Solution Mixture with equal amounts of Potassium Bichromate and Trichloroacetic acid 30~40 min. k1

Wash Running water → Distilled water 3 min.

Nuclear Stain Iron Hematoxylin 10 min. for every section k2

Stain enhancing 5~10 min.

2nd Mordant Solution Mixture with equal amounts of phosphotungstic acid-2.5% phosphomo-lybdic acid (only when using iron hematoxylin) 45~60 sec. , within 1 min. k3

Stain 0.75% Orange G Solution 10 times k4

1~2 min. for Iron-hematoxylin stain

Wash 1% Acetic Acid Solution I, II Wash gently for 2~3 sec.

Stain (cytoplasm) Ponceau Xylidine . Fuchsin Acid, Azophloxine Solution 20~30 min.

Wash 1% Acetic Acid Solution I, II Wash gently

Mordant Solution 2.5% 7~10 min.

Wash 1% Acetic Acid Solution I, II Lightly wash

Stain (connective tissue) Aniline Blue more than 3 min. k5

Wash 1% Acetic Acid Solution I, II Lightly wash k6

Differentiate/Dehydrate Isopropyl alcohol or 100% alcohol one-by-one k7

ClearXylene IXylene IIXylene III

2~3 min. each

Mount

k1: Prolonged treatment will reduce the quality of the aniline blue stain to some extent. The proper treatment time will depend on the thickness of the section.

k2: We recommend staining for a longer duration and differentiating with 0.5 to 1% acid alcohol. If possible, prepare agents I and II when performing the protocol.Double strength Carazzi’s hematoxylin can be used as a substitute but will require more time for staining compared to iron hematoxylin.

k3: The more effective the 1st mordant solution, the more red the iron hematoxylin will turn out. The 2nd mordant is used to maintain the dark hue, but prolonged treatment will easily diminish the quality of the aniline blue stain. Hence, do not treat the specimen with the 2nd mordant solution for more than one minute.

k4: You may skip the Orange G in this step and replace aniline blue with a mixture of aniline blue and Orange G for AZAN staining. Staining performance will remain virtually the same. Use undiluted aniline blue and Orange G solutions if you decide to employ this method. (0.8% Orange G is also acceptable.)

k5: Staining time for aniline blue will vary depending on the organ, condition and section thickness, so monitor the specimen carefully with microscopy. Treat the specimen with aniline blue again if staining performance is insufficient.

k6: Prolonged treatment to 1% acetic acid hydrate will reduce aniline blue quality. If this happens, prepare two tanks: wash the section in one tank and fix in the other. This will improve the balance between red and blue colorization.

k7: Differentiate each section carefully. Use the AZAN staining protocol as a reference. Isopropyl alcohol is appropriate for differentiating. If you believe you have stained for too long, use pure ethanol which has the effect of somewhat weakening the blue colorization.


StainsG

Masson’s Ttrichrome Stain G-3. Connective Tissue Staining

Staining Resultsl Nuclei: purplish black ~ purplish redl Collagen Fiber, Glomerular Basement Membrane, Reticular Fiber: bright blue l Mucus: bluel Fibrin: red ~ reddish orangel Basophils: bluel Eosinophils: redl Cytoplasm: pale red ~ purplish redl Red Blood Cell: orangish yellow ~ orangish red

Product Name Grade Package Size Wako Cat. No.Aniline Blue Solution for Pathology Research 500 mL 015-18045

0.8% Orange G Solution for Pathology Research 500 mL 159-02245

Ponceau Xylidine, Fuchsin Acid, Azophloxine Solution for Pathology Research 500 mL 166-19765

Azan Stain Azan stain is a method of staining collagen fibers selectively with aniline blue. At the same time, pathologic products such as hyaline degeneration and fibrin can be stained in different colors besides reticular fibers.

Deparaffinize

Xylene IXylene IIXylene III

5 min. each

100% Alcohol I100% Alcohol II100% Alcohol III70% Alcohol

2~3 min. each

Dealcoholize/Wash Running water → Distilled water Hydrate

Mordant Mixture with equal amount of Potassium Bichromate and Trichloroacetic acid 10 min. or 30 min. if the section does not stain red k1

Wash Running water → Distilled water 5 min.

Stain Azocarmine G Solution 5~6 hr. (more than 30 min.) at RT k2

Wash Distilled water Wash gently

Differentiate Aniline, Alcohol Perform quickly k3

Stop differentiation Acetic Acid, Alcohol 1 min. k4

Wash Running water → Distilled water Wash gently

Mordant 5% phosphotungstic acid more than 1 hr. or overnight k5

Wash Distilled water Wash Gently

Stain Aniline Blue, Orange G Solution 30 ~ 60 min. k6

Differentiate/Dehydrate Isopropyl alcohol or 100% alcohol one-by-one k7

ClearXylene IXylene IIXylene III

2~3 min. each

Mount

k1: This step is unnecessary if you are using Helly's or Zenker's fixative. Prolonged exposure will reduce the quality of the aniline blue stain.

k2: Stain thoroughly at room temperature. You can raise the temperature to about 60°C if you are in a hurry, but this will degrade the quality of the staining solution.

k3, 4: Differentiate each section individually because exposure to aniline/alcohol leads to faster decolorizing. Differentiate until you can distinguish the cytoplasm and the nuclei, and when the fibers become light red. Staining should be somewhat strong since decolorization will progress with exposure to tungsten phosphate in the next step. If you have stained properly you will not be able to differentiate individual component. Then you should place the section in tungsten phosphate for an extended length of time. This is a key to achieving a color that is not too dark and not too light, and will greatly affect the quality of the aniline stain. Instead of differentiating once, differentiate several times while making sure you stop the reaction with acetic alcohol in between rounds and you monitor progress with microscopy after washings.

k5: This is the mordant solution for aniline blue. Because new solution will result in strong decolorization, use an equal concentration mixture of new and old solution.

k6: Instead of using a mixture, perform Orange G staining and aniline blue staining independently. After mordanting, stain the section in a solution made by adding one drop of glacial acetic acid to 50 ml of 0.75% Orange G. Wash the section lightly with tap water, pass through distilled water and then transfer to azocarmine G-drifting solution. Do not add aniline blue to the Orange G solution. Compared to staining with a mixture, you should notice a prominent Orange G hue. The red blood cells should be yellowish and nuclei and cytoplasm should be brightly colored.

k7: Differentiation with aniline blue will determine whether the AZAN stain is acceptable. Be sure to replace the staining solution with ethanol immediately. Coloring will become dull if the section is passed through water. Wipe away solution from the back side of the slide glass and the surrounding area of the section. Tilt the slide glass and wash away staining solution using a pipette filled with isopropyl alcohol or pure ethanol. Lay the slide glass flat and apply alcohol. Shake the slide very gently and then decant. Repeat this alcohol rinse of the section until you are able to differentiate blue, red and intermediate color stains. You may perform this process in a staining dish if you have many sections, but be sure to replace the alcohol meticulously. If you want to repeat staining, decolorize using a low-concentration ammonia hydroxide solution.


Stai

nsG

Azan Stain G-3. Connective Tissue Staining

Staining Resultl Nuclei: Dark redl Fibrin: Redl Basophils: Bluel Eosinophils: Redl Collagen, glomerular basement membrane, reticular fibers, hyaline material, mucus: Bright blue

Staining Resultl Elastic Fibers, HBs antigens, Cartilage matrix: bluel Nuclei: Redl Background: pale pink

n�Aniline Blue · Orange G Solution Composition

Victoria Blue Staining Elastic fibers and HBs antigens stain blue. Oxidation and reduction is necessary for pretreatment when staining HBs antigens.

n�Azocarmine G Solution Composition

Aniline Blue .............................0.5 gOrange G ......................................2 gDistilled Water .....................100 mLAcetic Acid ..................................8 mL

Azocarmine G .......................0.1 gDistilled Water .....................100 mLAcetic Acid ..................................1 mL

Product Name Grade Package Size Wako Cat. No.Aniline Blue · Orange G Solution for Pathology Research 500 mL 012-18055

Product Name Grade Package Size Wako Cat. No.Victoria Blue Solution for Pathology Research 500 mL 223-01475

Product Name Grade Package Size Wako Cat. No.Azocarmine G Solution for Pathology Research 500 mL 019-18065

Deparaffinize

Victoria Blue Solution 30 min.~ 1 hr.

70% Ethanol a few times

Wash with running water 3 min.

Kernechtrot (Nuclear fast red) 5 min.

Wash with running water 1 min.

70% Ethanol 1 time

95% Ethanol 2 times

100% Ethanol 3 min. × 3 times

Clear/Mount


StainsG

G-4. Staining of Fibrins and Glial Tissues

Phosphotungstic Acid Hematoxylin Staining The product can stain neuroglia fiber and collagen fiber in different colors, and is also used for staining muscle fiber and fibrin.It is useful for discriminating neuroglia fiber and connective fiber in central inflammatory disease and brain tumor and for observing the extent of metastasis.

G-5. Intracellular Inorganic Substance Staining

Berlin Blue Staining This staining is used for staining trivalent iron ion in tissues, especially as hemosiderin staining. Hemosiderin is a yellowish- or brownish-red dye originated from hemoglobin, and found in the tissues and cells after bleeding.

Tissue Section

Deparaffinize

Saturated Picric Acid 15 min.

Wash with water

Lugol’s Solution 15 min.

0.5% Hypo 3 min.

Wash with water 5 min.

PTAH Staining Solution62°C 90 min.

RT 30 min.

100% Alcohol

I 3 min.

II 3 min.

III 3 min.

Clear/Mount

CompositionHematoxylin ............................................1 gPhosphotungstic Acid ..................20 gDistilled Water ..............................1,000 mL

Kit Components2% Hydrochloric Acid..........................................500 mL × 1 bottle2% Potassium Ferrocyanide ..........................500 mL × 1 bottle

Preparation of Staining Solution: Mix equal amount of each solution just before use.


Phosphotungstic Acid Hematoxylin Solution for Pathology Research100 mL 167-15611500 mL 169-15615

Tissue Section

Deparaffinize/Wash with water

Thoroughly wash with distilled water

Berlin Blue Staining Solution (at RT) 15 ~ 30 min.

Thoroughly wash with distilled water

Nucleus Staining

Wash with water


Product Name Grade Package Size Wako Cat. No.Berlin Blue Staining Set for Pathology Research 1 set 296-21541

Human Spleen


Stai

nsG

G-6. Cytological Staining

Papanicolaou Staining Papanicolaou stain is for cytological diagnosis and is an essential staining method for the early detection and definitive diagnosis of cancer.

Principle of Papanicolaou stainNucleus Staining

Hematoxylin stains nuclei. Hematoxylin is converted by an oxidizing agent to hematein, which upon addition of alum will form a chelate bond with aluminum, resulting in aluminum rack. This is positively charged and has staining ability, and upon binding to the negatively charged phosphate group of a nucleic acid will turn it dark purple.

n�Papanicolaou-Gill’s Hematoxylin (Gill-5) Stain Solution Composition

n�Other Papanicolaou Staining Solution Papanicolaou EA100, OG100 and Papanicolaou-Hematoxylin Stain Solution are cytoplasmic nuclear stain solutions of Papanicolaou’s stain commonly used in cyto-logical diagnosis. These have almost the same stain pattern as the conventional one but have been improved so as to retain stain accuracy for a long time. These were prepared according to a protocol developed by Tenjin, et al.

Hematoxylin .......................................5 gDistilled water .............................. 730 mL

Aluminum sulfate ........................................44 gSodium Iodate ..........................................0.52 g

Ethylene glycol ...................................... 250 mL

Fix (95% Alcohol) 30 min.

Hydrate (80, 70, 50% Alcohol)


Nucleus Stain (Gill's Hematoxylin) 1~2 min.

Differentiate


70% Alcohol5~10 times

1% Hydrochloric Acid-Alcohol


Dehydrate (95% Alcohol)

Cytoplasm staining (OG-6) 3 min.

Differentiate 100% Alcohol × 2

Cytoplasm staining (EA-50) 3 min.

Differentiate 100% Alcohol × 2

Dehydrate (100% Alcohol × 4)

Clear (Xylene × 5)

Mount (Softmount: Refer to p28)

Product Name Grade Package Size Wako Cat. No.Papanicolaou-Gill's Hematoxylin (Gill-5) Stain Solution for Pathologic Cytodiagnosis Research 500 mL 167-19795

Fix 95% Alcohol 30 min.

Stain

95% Alcohol 10 times




Wash with water Wash gently

Hematoxylin Stain Solution 3 min.

Wash with water Wash gently

0.5% Hydrochloric Acid Apply suitably

Wash with water 5 min.





Stain

OG100 Stain Solution 1 min.



EA100 Stain Solution 2 min.






Xylene 10 times

Xylene 10 times

Xylene 10 times

Mount

Product Name Grade Package Size Wako Cat. No.Papanicolaou, Hematoxylin Stain Solution for Pathologic Cytodiagnosis Research 250 mL 168-18941

Papanicolaou EA100 Stain Solution for Pathologic Cytodiagnosis Research 250 mL 164-18921

Papanicolaou OG100 Stain Solution for Pathologic Cytodiagnosis Research 250 mL 161-18931

Adenocarcinoma cells of Sputum


StainsG

G-7. Blood Cell Staining

Staining Results

n�Giemsa Stain Solution Staining Protocol

n�May-Grünwald’s Eosin Methylene Blue Solution (MGE solution) The solution is used for staining of blood cell components such as erythrocytes, basophil granules, and neutrophil granules in blood and pathological tissues.

n�Wright Stain Solution Staining Protocol 1 (Wright Stain)

n�May-Grünwald Stain Solution Staining Protocol (May-Grünwald, Giemsa Stain)

① Fix: Add a few drops of methanol ..........................................................abt. 30 sec.② Preparation of Giemsa Staining Solution: Add Giemsa Staining

Solution at a ratio of 1 to 1.5 drops per 1 mL of water. (2 ~ 3 mL of the prepared Giemsa Staining Solution will be required for each specimen.)

<Note> Dilute the Giemsa Staining Solution just before use. This is because if the solution is left standing, azure B and eosin Y molecules will bond, resulting in a loss of staining performance.

③ Staining: Apply the prepared staining solution to the painted side .....15 ~ 30 sec.④ Wash with water ...............................15 ~ 30 sec.⑤ Dry

① Fix, Stain: Add 10 ~ 15 drops of Wright Stain Solution ............... 2 ~ 3 min.② Add 10 ~ 15 drops of phosphate buffer onto the 1. and mix ..... 4 ~ 7 min.

③ Wash with running water .......15 ~ 30 sec.④ Dry

Staining Protocol 2 (Wright-Giemsa Stain)

① Fix & Stain: Add 10 ~ 15 drops of Wright Stain Solution ........................... 2 ~ 3 min.② Add 10 drops of phosphate buffer ............................................................................... 2 ~ 3 min.③ Wash: Pour the prepared Giemsa Staining Solution from above ............15 ~ 30 sec.④ Stain: prepared Giemsa Staining Solution ........................................................ 10 ~ 15 min.

⑤ Wash with running water⑥ Dry<Note> Wright’s one step stain provides

brightly colored granules, but also drably stained nuclei.

① Fix & Stain: Add 10 drops of May-Gruenwald Stain Solution ...........................2 ~ 3 min.② Add 10 drops of phosphate buffer ...................2 ~ 3 min.③ Wash with prepared Giemsa Staining Solution .....2 ~ 3 min.

④ Wash: prepared Giemsa Stain Solution .......10 ~ 15 min.⑤ Wash with running water⑥ Dry

Deparaffinize / Wash with water

Acidic treatment with Decalcifying Solution A 5 ~ 30 min.

Wash with distilled water 5 min.

MGE Solution 10 min.

Tripling Diluted MGE Solution*1 30 min.

Wash with water (Wash off the excess pigment)

Giemsa Stain 2 hr.

Wash with distilled water 10 times

Differentiate with Acetic Acid Solution *2 30 times


Wash with distilled water 1 min.

Absolute alcohol 30 times

Clear/Mount

Product Name Grade Package Size Wako Cat. No.Giemsa Stain Solution for Pathologic Cytodiagnosis Research 250 mL 079-04391

May-Grünwald Stain Solution for Pathologic Cytodiagnosis Research 250 mL 131-12811

Wright Stain Solution for Pathologic Cytodiagnosis Research 250 mL 238-01541

Product Name Grade Package Size Wako Cat. No.May-Grünwald's Eosin Methylene Blue Solution for Pathology Research 500 mL 138-11645

UsageAdd this solution into methanol, dissolve it by warming and mixing for 1 hour at 40°C. After slowly-cooling, filter it.

Staining ResultsNuclei: reddish purpleAcidophilic granule: red or reddish brownNeutrophil granule: reddish purpleBasophilic granule, platelet: BlueErythrocyte: pale red

Nuclei: RedCytoplasm: Blue ~ pale blue

Acidophilic granule: RedBasophilic granule: purplish blue ~ blue

Erythrocyte: pinkish red

k�1: Dilute with distilled waterk�2: Prepare the solution with

distilled water (200 mL) + acetic acid (3 drops)

Leukemic lymph node


Stai

nsG

G-8. Hard Tissue Staining

TRAP/ALP Stain Kit Normal bone metabolism is based on the balance between bone formation by osteoblasts and bone resorption by osteo-clasts. When the balance is disturbed and bone resorption by osteoclasts is abnormally increased, bone mass is reduced leading to osteoporosis. Therefore, various researches have been carried out to understand the mechanism of osteoclast and osteoblast metabo-lism, and to utilize this knowledge for the treatment of the diseases and for the development of effective drugs.Today, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) are known as marker enzymes for osteoblasts and osteoclasts, respectively, and these enzymes are used as one of the markers showing the presence of osteoblasts and osteoclasts in tissue sections or cultured cells.This kit enables you to examine the state of differentiation of bone cells and the cell distribution in bone tissues by observation of the stained images of osteoblasts and osteoclasts in the tissues and cultured cells using ALP/TRAP enzyme activities in the tissue sections and cultured cells.By studying the stained image of osteoblasts and osteoclasts, you can track their distributions and differentiation status in bone tissue.

Featuresl �In use, just mix two solutions for the preparation of the colorimetric

substrate solution required for staining of acid phosphatase enzyme activity. (Three solutions in case of the evaluation of tartrate-resistant acid phosphatase)

l �Simple steps for staining of alkaline phosphatase enzyme activity by using ALP substrate soln. (premixed).

l �Double staining can be performed, reddish-purple for active region of acid phosphatase, and blue to bluish brown for alkaline phosphatase.

l �Usable in cultured cells and bone tissue sections (GMA resin embedded section).

Kit Components① Sodium tartrate soln. (×10) .......................................................................3 mL × 1 bottle② TRAP substrate soln. A ............................................................................30 mL × 1 bottle③ TRAP substrate soln. N .......................................................................... 0.3 mL × 1 bottle④ Nuclear stain soln. ........................................................................................10 mL × 1 bottle⑤ ALP substrate soln. (premixed) ..........................................................30 mL × 1 bottle<Note> This kit contains enough reagent to stain an equivalent of five 24-well culture plates,

six 96-well culture plates, or 60 bone tissue slides (when using 500 μL per slide).

Activity Staining of TRAP/ALP in Cultured Cells

Product Name Grade Package Size Wako Cat. No.TRAP/ALP Stain Kit for Pathology Research 60 tests 294-67001

Epiphyseal plate chondrocytes of the vertebral disk

Primary trabecula

Bone marrow cells

Trabecula

Epiphyseal plate chondrocytes

OsteoblastsOsteoclasts

k Osteoclasts stain red with TRAP staining solutionk �Chondrocytes, intercellular membranes and the cell

membranes of osteoblasts stain brownish-red with ALP staining solution.

k �Nuclei of various cells stain bluish green with nuclear staining solution.

Activity Staining of TRAP in RAW 264 cells

Activity Staining of ALP in MC 3T3-E1 cells


StainsG

G-9. Other StainsStain Product List


Acridine Orange (CI 46005) Practical Grade 5 g 014-0894125 g 012-08942

Acridine Yellow - 25 g 018-00742Aniline Blue · Water Soluble [Water Blue] - 25 g 016-21302Auramine (CI 41000) Wako 1st Grade 10 g 013-04871Bismarck Brown (CI 21000) Wako 1st Grade 25 g 028-01902

Carmine (CI 75470) Wako Special Grade 5 g 035-0137125 g 033-01372

Chlorazol Black E for Protozoan Kohn's Staining 25 g 038-17432Congo Red (CI 22120) JIS Special Grade 25 g 032-03922

Crystal Violet (CI 42555) JIS Special Grade 25 g 031-04852for Pathology Research 25 g 038-17792

4',6-Diamidino-2-phenylindole Dihydrochloride n-Hydrate [DAPI] for Biochemistry

5 mg 043-1880410 mg 049-18801

100 mg 045-18803

Eosin Y (CI 45380) Wako Special Grade 25 g 058-00062for Pathology Research 25 g 056-06722

Erythrosine (CI 45430) for Microscopy 25 g 053-00252

Fast Blue RR Salt (CI 37155) - 5 g 061-0371125 g 069-03712

Fuchsin Acid (CI 42685) Practical Grade 25 g 061-01332

Fuchsine Basic (CI 42510) Wako Special Grade 10 g 066-0058125 g 064-00582

Gentian Violet R [Methyl Violet] Wako Special Grade 25 g 136-03272

Hematoxylin Monohydrate (CI 75290) for Cell Staining 5 g 088-0746125 g 086-07462

Indigo Carmine (CI 73015) for Pathology Research 25 g 093-04732Light Green SF Yellowish (CI 42095) - 25 g 124-04632Malachite Green Oxalate for Pathology Research 25 g 131-13732Metanil Yellow Wako Special Grade 25 g 137-01502

Methyl Green - 10 g 134-1390125 g 132-13902

Methyl Violet B (CI 42535) Wako Special Grade 25 g 130-03292

Methylene Blue Tetrahydrate (CI 52015) for Vital Staining 25 g 137-06982500 g 131-06985

Neutral Red (CI 50040) Wako Special Grade 25 g 140-00932Nile Blue Hydrogensulfate for Pathology Research 25 g 141-06822

Nile Red for Pathology Research 25 g 144-08811100 mg 140-08813

Oil Red O for Pathology Research 25 g 154-02072

Orange G (CI 16230) Wako 1st Grade 25 g 150-01832for Pathology Research 25 g 152-02252

Orcein Practical Grade 1 g 157-009435 g 151-00941

Phloxine B (CI 45410) Wako Special Grade 25 g 166-02072

Pyronine Y (CI 45005) Practical Grade 5 g 164-1158125 g 162-11582

Safranine (CI 50240) Wako Special Grade 25 g 196-00032

Sirius Red (CI 35780) for Pathology Research 10 g 196-1620125 g 194-16202

Sudan 1 (CI 12055) Wako 1st Grade 25 g 195-04382Sudan 2 (CI 12140) Wako Special Grade 25 g 198-06072Sudan 3 (CI 26100) Wako Special Grade 25 g 192-04392Sudan 4 (CI 26105) Wako 1st Grade 25 g 194-07652Sudan Black B (CI 26150) Wako 1st Grade 25 g 192-04412Thioflavin T (CI 49005) - 25 g 202-01002

Thionin Acetate for Pathology Research 5 g 208-1861125 g 206-18612

Victoria Blue B (CI 44045) Wako 1st Grade 25 g 228-00222Victoria Blue 4R (CI 42563) Wako 1st Grade 25 g 223-00772Wright Stain, Powder Wako Special Grade 10 g 235-00191


Mou

ntin

g Re

agen

tH

H. Mounting Reagent

H-1. SoftmountA xylene-free oil-soluble mounting ReagentSoftmount is a very safe high-performing mounting reagent that does not contain hazardous xylene.Adjust Softmount viscosity by diluting with Lemosol® A.

Features

I. Antigen Retrieval Reagent

I-1. ImmunoSaverImmunoSaver is an antigen retrieval reagent for formalin-fixed tissues. Formalin-fixed tissues are known to form a methylene bridge between proteins, masking the antigenic site and reducing antigenicity. The antigenicity can be recovered by using this product. Conventional methods required consideration in a variety of conditions. However, this product can activate a wide range of antigens under the same conditions.

Product Name Grade Package Size Wako Cat. No.ImmunoSaver for Immunohistologic Staining 25 mL 097-06192

Featuresl Can activate antigenicity in tissue fixed with formalinl Can improve immunostaining performancel �Can activate a wide range of antigens under the same conditions

Formulation10% Citraconic Acid Sodium Solution

Data

Procedure① Dilute ImmunoSaver 200 times with distilled

water or deionized water, and then heat to 98°C.② Place the deparaffinized tissue sections into the

solution and incubate for 4 5 minutes at 98°C.③ Wash 3 times in PBS for 5 minutes and then

perform immunostaining.

l Fast drying performance is equal to xylene mountantsl Reduced odor and toxicityl �Contains a UV absorber and an antioxidant (to prevent

deterioration or change in color)

l �Can be used for H&E staining, special-purpose staining and immunostaining

l �Features superior extensibility and transparency during mounting

l Refractive Index: abt. 1.50 (20°C)

Product Name Grade Package Size Wako Cat. No.Softmount for Pathology Research 250 mL 192-16301


Apathy's Mounting Media, Soluble for Pathology Research 100 mL 010-13811

Canada Balsam Wako 1st Grade25 g 034-01042

100 g 036-01041500 g 038-01045

Antig

en R

etriev

al Re

agen

tI

200μm

50μm

200μm

50μm

1) Treat formalin-fixed paraffinized sections of the human pancreas with ImmunoSaver. 2) Wash once with RBST (PBS containing 0.2% Triton X-100), and twice with PBS. 3) Perform blocking at room temperature for one hour. 4) Rinse lightly with PBS and react with PBST-diluted primary antibody (rabbit anti-SNAP-25 at 1:8,000)

at 4°C for one night. 5) Wash once with PBST and twice with PBS. 6) React with biotinylated secondary antibody for one hour at room temperature. 7) Wash once with PBST and twice with PBS. 8) React with peroxidase-labeled avidin (diluted with PBS at 1:400) for one hour at room temperature. 9) Wash once with PBST and twice with PBS.10) Produce color in the matrix with DAB11) Wash three times with distilled water, using five minutes per wash.12) Stain with Mayer's acid hemalum for one to three minutes.13) After washing in running water, rinse two to three times with distilled water and then differentiate with

70% ethanol.14) Clear and mount.

Human Pancreas SNAP-25


Gene-Related Kits

J

J. Gene-Related Kits

J-1. Apoptosis in situ Detection Kit Wako

Apoptosis Detection Kit Using the TUNEL MethodThis kit is for detection of apoptotic cells. It uses the TUNEL method for detection. The kit contains main reagents ready for use and is suitable for quick and easy analysis. It can be used with paraffin-embedded sections, cultured cells (neutral buffered formalin fixed), and cryosections.

Featuresl Includes main reagents ready for use and no more time-consuming preparation.l Needs only about 2 hours for the entire procedure.l Low Background and Clear Signal.

Kit ContentsProtein Digestion Enzyme............................1 mL × 1 vialTdT ..............................................................................40 μL × 1 vialTdT Substrate Solution .............................4.4 mL × 1 vial100 × POD-Conjugated Antibody .......44 μL × 1 vialDNase I .......................................................................4 μL × 1 vial10 × DNase I Reaction Buffer ................40 μL × 1 vial

ApplicationTransplanted breast cancer in a mouse treated with a-Mangostin (× 200)

Product Name Grade Package Size Wako Cat. No.Apoptosis in situ Detection Kit Wako for Apoptosis Research 40 tests 293-71501


Apoptosis Ladder Detection Kit Wako for Apoptosis Research24 lanes 297-7140196 lanes 293-71403

DAB Tablet (DAB. 4HCl 10 mg/Tablet) for Biochemistry50 tablets 049-22831

100 tablets 045-22833

DAB Tablet (DAB. 4HCl 5 mg/Tablet) for Biochemistry50 tablets 040-27001100 tablets 046-27003

DAB TRIS Tablet, pH 7.6 for Biochemistry 50 tablets 047-27011

<Note> The kit does not contain a chromogenic substrate. We recommend GenWay’s two-component liquid substrate, DAB Immunohistochemistry Substrate (#45-053-150038).

Apoptosis of carcinoma cells due to antitumor effect by a-Mangostin was confirmed .(Data was provided by Professor Masaaki Shibata, Osaka Health Science University. )

Control group a-Mangostin group


Gene

-Rel

ated

Ki

tsJ

J-2. DNA Isolator PS KitDNA Isolation from Paraffin-embedded tissue sectionsThis kit can isolate DNA from paraffinized sections in a short length of time.

J-3. DNA Isolator PS-Rapid ReagentDNA extraction from paraffin-embedded sections in 20 minutes.

Product Name Grade Package Size Wako Cat. No.DNA Isolator PS Kit for Genetic Research 100 tests 295-52401

Product Name Grade Package Size Wako Cat. No.DNA Isolator PS-Rapid Reagent for Genetic Research 100 tests 291-56401


Ethanol (99.5) for Molecular Biology100 mL 052-07221500 mL 054-07225

2-Propanol for Molecular Biology100 mL 166-21671500 mL 168-21675

Featuresl �No de-paraffinization and proteolytic digestion are

necessary.l DNA Isolation with two simple steps in 20 minutes.l Useful for Amplification of DNA less than 500 bp.l Only a minute amount of PCR inhibitor is contaminated.

Kit ComponentsDNA Isolation Solution ......................10 mL × 5 drop-style tubes(One drop from the eye drop-style tube contains about 30 μL of solution)

DataAmplification of the b-globin gene using DNA isolated from paraffinized sections of various tissue specimens as templates.

Featuresl DNA Isolation within 90 minutesl �Minimal DNA Damage because the entire protocol is performed

with only one tube.l Safe Operation: No phenol or chloroform requiredl Isolated DNA can be used immediately for PCR.

Kit ContentsEnzyme reaction solution ..................................................2 mL × 1 vialEnzyme Activator .................................................................34 mg × 1 vialProtease .....................................................................................2.2 mg × 1 vialAccelerator for DNA Precipitation ..........................0.5 mL × 1 vialDilution Solution for DNA Amplification .............0.5 mL × 1 vial(Deparaffinizing reagent such as Lemosol® A (Wako Catalog No. 120-04411) is necessary additionally. (See page #9))

Data

Amplification of the b-globin gene using DNA isolated from paraffinized sections of various tissue specimens as a template.

a: DNA Isolator PS Kitb: conventional method3% Agarose gel

amplified band (110 bp)

Marker

Stomach Colon Colon Cancer Muscle Lymp-

homaPanc-reas Vesica Kidney

This kit

Liver PancreasCompany A This kit Company A This kit

Heart ColonCompany A This kit Company A This kit

Stomach KidneyCompany A This kit Company A

M: f X174/Hae III 1: b-globin (205 bp) 2: b-globin (325 bp) 3: b-globin (408 bp)


Gene-Related Kits

J

J-4. ISOGEN PB KitRNA Extraction Kit from Paraffin-Embedded Tissue SectionsThe ISOGEN PB Kit is for extracting RNA from paraffin-embedded tissue sections.RNA can be extracted in 2 hours by the following simple manipulations:

Deparaffinization → Proteinase K treatment → RNA extraction

Product Name Package Size Wako Cat. No.ISOGEN PB Kit <manufactured by Nippon Gene Co., Ltd> 20 tests 315-06421

<Related Products>Product Name Package Size Wako Cat. No.

ISOGEN-LS <manufactured by Nippon Gene Co., Ltd>10 mL 317-02623

100 mL 311-02621

Use of Extracted RNAl Detection of RNA virus by RT-PCRl Detection of mRNA by RT-PCRl �Checking the preservation of RNA in paraffin-embedded tissue

sections, etc.

Kit Contents (for 20 tests)Proteinase K (20 mg/mL) ................. 100 μL × 1Extraction Buffer ................................. 3 mL × 1ISOGEN-LS ....................................... 10 mL × 1Ethachinmate ..................................... 60 μL × 1Deoxyribonuclease (RT Grade) .......... 20 μL × 110 × DNase (RT Grade) Buffer II ...... 100 μL × 1Stop Solution (RT Grade) ................. 100 μL × 1DEPC treated Water ......................... 500 μL × 2

Reagents necessary in addition to the kitLemosol® or Lemosol® A, Ethanol, Chloroform, Isopropanol

ApplicationDetection of Gapd gene by RT-PCRRNA was extracted using 10 μm × 4 sheets of paraffin-embedded tissue section from mouse submaxillary gland in accordance with the protocol. The 500 ng of obtained RNA was treated with DNase by the protocol, followed by detection of mouse Gapd gene exon 5 (258 bp) by RT-PCR.

Lane 1: OneSTEP Marker 5 (fX174/Hinc II digest)Lane 2: No templateLane 3: Template = RNA without reverse transcription reactionLane 4: Template = RNA with reverse transcription reactionLane 5: OneSTEP Marker 5 (fX174/Hinc II digest)

RNA Extraction ProtocolParaffin-Embedded Tissue Sections❋

RNA

Lemosol® or Lemosol® ALeave for 1 min. at RT

100% EthanolCentrifuge (12k × g, 2 min. at RT)

100% EthanolLeave for 1 min. at RTCentrifuge (12k × g, 2 min. at RT)

70% EthanolCentrifuge (12k × g, 5 min., at 4℃)

Dry

Extraction Buffer

Proteinase K Heat for 15 min ~ 16 hr. at 50℃.

ISOGEN-LSLeave for 5 min. at RT.ChloroformLeave for 3 min. at RT.Centrifuge (12k × g, 15 min. at 4℃)

Ethachinmate IsopropanolLeave for 5 ~ 10 min. at RTCentrifuge (12k × g, 10 min., at 4℃)

De-paraffinization

Proteinase KR

NA Extraction

Precipitation

Precipitation

Aqueous phase

Precipitation

❋ : In case that 4 sections (< 25mm2 × 10µm) are used.


Paraffi

n-emb

edde

d Tis

sue Se

ction

sK

Product Name Grade Package Size Wako Cat. No.HistoMap Mouse Normal Cerebellum, Paraffin Embedded Tissue for Pathology Research 10 sheets 089-09571HistoMap Mouse Normal Cerebrum, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 082-09561HistoMap Mouse Normal Heart, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 087-09491HistoMap Mouse Normal Kidney, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 081-09531HistoMap Mouse Normal Liver, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 086-09581HistoMap Mouse Normal Lung, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 087-09511HistoMap Mouse Normal Pancreas, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 084-09521HistoMap Mouse Normal Rectum, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 080-09481HistoMap Mouse Normal Spleen, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 080-09501HistoMap Mouse Normal Stomach, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 088-09541HistoMap Mouse Normal Testis, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 085-09551

Product Name Grade Package Size Wako Cat. No.HistoMap Rat Normal Cerebellum, Paraffin Embedded Tissue for Pathology Research 10 sheets 080-09621HistoMap Rat Normal Cerebrum, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 081-09651HistoMap Rat Normal Heart, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 087-09631HistoMap Rat Normal Kidney, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 084-09641HistoMap Rat Normal Liver, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 083-09611HistoMap Rat Normal Lung, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 085-09671HistoMap Rat Normal Pancreas, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 089-09691HistoMap Rat Normal Rectum, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 088-09661HistoMap Rat Normal Spleen, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 082-09681HistoMap Rat Normal Stomach, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 086-09601HistoMap Rat Normal Testis, Paraffin Embedded Tissue Section for Pathology Research 10 sheets 082-09701

K. Paraffin-embedded Tissue Sections

K-1. HistoMap SeriesHistMap series are paraffin-embedded tissue sections from normal rat or mouse fixed for 3 days in 10% neutral buffered formalin and can be used for HE staining, immunostaining and other types of staining protocols.

Section photoHE Staining

Paraffin-embedded Tissue Section from mouse

Paraffin-embedded Tissue Section from rat

Wako Chemicals USA, Inc.www.wakousa.comToll-Free (U.S. only): 1-877-714-1920Head Office (Richmond, VA): Tel: 1-804-714-1920/ Fax: 1-804-271-7791Los Angeles Sales Office (Irvine, CA):Tel: 1-949-679-1700 / Fax: 1-949-679-1701Boston Sales Office (Cambridge, MA):Tel: 1-617-354-6772 / Fax: 1-617-354-6774

Wako Pure Chemical Industries, Ltd.www.wako-chem.co.jp

1-2, Doshomachi 3-ChomeChuo-Ku, Osaka 540-8605, JapanTel: 81-6-6203-3741Fax: 81-6-6203-1999

Wako Chemicals GmbHwww.wako-chemicals.deEuropean Office: Fuggerstra e 12, D-41468Neuss, GermanyTel: 49-2131-311-0Fax: 49-2131-311100

l ICR mouse (SPF animal) l Age, Sex: 10 week-old, male l Thickness: abt. 3 μm l Fixing: 10% neutral buffered formalin

l F344/DuCr1j (SPF animal) l Age, Sex: 10 week-old, male l Thickness: abt. 3 μm l Fixing: 10% neutral buffered formalin

n Listed products are intended for laboratory research use only, and not to be used for drug, food or human use.n Please visit our online catalog to search for other products from Wako : www.e-reagent.comn This brochure may contain products that cannot be exported to your country due to regulations.n Bulk quote requests for some products are welcomed. Please contact us.

mouse

rat

Please remove the protective seal on the product before use.

testicle lung kidney stomach

13315GK

Download - Pathology Research 2013 - wako-chem.co.jp

Top Related