Tissue Stains

(H&E) (PAP)

Prepared by M Hlasek March 2017

Reviewed February 2020

Aims of staining

Commonly used medical process in the medical diagnosis of tumors

Technique used to enhance contrast in samples

Make the cell structure visible

Show variation in structure

Indicate the chemical nature of tissue entities

Staining methods

Haematoxylino Three main types:

Alum

Iron

Tungsten

o Other:

Lead

Molebdenum

Haematoxylin without mordant

These haematoxylins are named as such because of the mordant that is used.

Alum Haematoxylin

The mordant contains aluminium egPotassium aluminium sulphate in Mayer’s Haematoxylin.

Disadvantage: very sensitive to acid solutions

Iron HaematoxylinHere the mordant is an iron salt eg ferric chloride or ferric ammonium. These salts acts also as the oxidising agent.

Disadvantage: Over oxidise and “ripen” very quickly

Tungsten HaematoxylinThe haematoxylin can be ripened chemically with potassium permanganate or left to ripen in sunlight.

Disadvantage: only Mallory’s phosphotungstic acid haematoxylin is the only one widely used

MordantIs a substance, typically an inorganic oxide, that combines with a dye or stain and thereby fixes it in a material/tissue section.

Ionic bonding / Coulombic attractions

Acid and basic dyes, and other ionic reagents, including inorganic salts

Hydrogen bonding

Is a dye-tissue attraction arising when a hydrogen atom lies between two

electronegative atoms

(e.g. oxygen or nitrogen)

Van der Waals forces

Intermolecular attractions as dipole-dipole, dipole-induced dipole and dispersion forces. These occur between all reagents and tissue

substrates.

Van der Waals Forces cont.

Covalent bonds Between tissue and stain also occurs, which bonds may be regarded merely as another source of stain-tissue affinity

Covalent bonds cont.

Hydrophobic interaction Staining systems using aqueous solutions of dyes or other organic reagents; enzyme substrates for example

Hydrophobic interaction

Histology classification of dyes

Basic dyes – these are cationic dyes and will stain anionic or acidic materials (e.g. the phosphates in nucleic acid) in the tissue e.g. Methylene Blue, Safranin, Basic fuchsin.

Acidic substances that stain with basic dyes are termed basophilic

Acidic dyes - these are anionic dyes and will stain cationic or basic groups in tissue such as amino groups e.g. Picric acid, Eosin, Acid fuchsin.

Substances that stain with acid dyes are called acidophilic

Histology classification of dyes continue

Neutral Dyes- simply compounds of basic and acidic dyes. Such dye complexes will stain both nucleus and cytoplasm e.g. Romanowsky stains, EosinY or Eosin B

Amphoteric dyes - have both anionic and cationic groups, but on the same ion. Such dyes stain either the nucleus or the cytoplasm if conditions are appropriate e.g. Celestine blue B, Acid fuchsin

Natural dyes – dye substances extracted from natural sources e.g. Haematoxylin from the logwood of a tree.

Important dyes in histology Nuclear stains

Haematoxylin

Carmine and Carminic acid

Methylene blue

Neutral Red and Safranine O

Methyl green

Toluidine blue

Cytoplasmic stains

Eosin

Methyl blue and aniline blue

Fast green FCF and Light green SF

Orange G

Routine stains

Haematoxylin and Eosin (H &E Stain) for histology

Papanicolaou stain (PAP Stain) for cytology

Principle of the H&E Stain The staining method is based on the

chemical attraction between tissue and dye. Charges on the dye and tissue are opposite and therefore attract (Van der Waals forces or Ionic bonding)

The Nuclei is acidic (-ve charges) and reacts with haematoxylin a basic dye

The cytoplasm or connective tissue are basic (+ve charges) components reach with Eosin an Acid dye.

Method H&E Staina. Scott’s Tap Water Sodium hydrogen carbonate 3.5gm/17.5gm Magnesium sulphate 20gm/100 gm

Tap water 1000ml/ 5000mlL

b. 1% Acid alcohol Ethanol 70 ml

Distilled water 30 ml Use 99ml of above, add HCL 1ml

OR Ethanol 3500ml

Distilled water 1500ml Use 4950ml of above, add HCL 50ml

H&E stain continuec. Eosin/Phloxine solution

1% Eosin Stock Solution

Eosin Y 1gm

Distilled water 100ml

1% Phloxine Stock Solution

Phloxine B 1gm

Distilled water 100ml

Eosin/Phloxine Working Solution

1% Eosin stock solution 80ml/380ml

1% Phloxine solution 40ml/160ml

Distilled water 120ml/480ml

H&E continueMethod

Dewax and hydrate sections to distilled water

Stain nuclei with the alum haematoxylin for 5-6 minutes

Rinse in running tap water

Differentiate with 1.0% acid alcohol for ±2 seconds (if staining regressively)

Rinse in tap water

Blue in Scott's tap water for 1minute OR blue in running tap water for 5 minutes

Rinse well in tap water

Counterstain with Eosin for 1- 2 minutes

Rinse well in tap water

Dehydrate, clear and mount.

H&E results

Collagen Pale pink

Muscle Deep pink

Acidophilic cytoplasm Red

Basophilic cytoplasm Purple

Nuclei Blue

Erythrocytes Cherry red

Calcium deposits Blue

Bacteria Blue

Mucin Pale blue/Grey

Cartilage Pale blue/grey

H&E results continue

H&E Troubleshoot

Special stains vs H&E

Papanicolaou Stain

Principle of the PAP stain

The pap stain is a special stain for keratin that is capable of detecting minimal or focal evidence of squamous cell differentiation.

As cells keratinized, the glassy cytoplasm progress from blue-green (abundant free ribosomes and prekeratin) to intensely orange (true keratin).

Principle of the PAP stain

Papanicolaou stain (also Pap stain) is a multi-chromatic staining histological technique histological technique.

Pap stain involves five dyes in three solutions:

A nuclear stain, haematoxylin, is used to stain cell nuclei.

OG-6 counterstain. The Orange G is used to stain keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.

EA (Eosin Azure) counterstain, comprising of three dyes; the number denotes the proportion of the dye e.g. Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells.

Light Green SF yellowish stains the cytoplasm of all other cells.

Bismarck brown Y stains nothing and in contemporary formulations it is often omitted.

Method of PAP staina. Scott’s tap water

Sodium Hydrogen Carbonate 3.5gm OR 17.5gm

Magnesium Sulphate 20gm OR 100gm

Tap Water 1000ml OR 5000ml

b. 95% Ethanol

Ethanol 50ml OR 5ml

DDW 950ml OR 95ml

c. Solutions OG-6 and EA-50 (commercial)

PAP stain continueProcedure 2 (Modified Pap Procedure): Fix in 95% Ethanol 15 minutes Rinse in 2 changes DDW - 10 dips each Stain in Gill’s Haematoxylin for 2 minutes Rinse in DDW - 10 dips Blue in Scott's tap water for 1 minute Rinse in 2 changes 95% Ethanol - 10 dips Stain in OG-6 stain for 5-7 minutes Rinse in 3 changes 95% Ethanol - 10 dips Stain in EA-50 (or EA-65 stain) for 6-10 minutes Rinse in 3 changes 95% Ethanol - 20-30 dips Rinse in Absolute ethanol - 10 dips Clear in xylene, mount

Results

Cell nuclei Crisp blue to black

Cells (high content of keratin) Yellow/orange

Glycogen stains Yellow

Superficial cells Pink to orange

Intermediate & para-basal cells Turquoise green to blue

Metaplastic cells Blue/green and pink at once.

Results continue

Results continueA. REGRESSIVE - Differentiator

Haematoxylin stain is applied Tissue is overstained with haematoxylin Differentiator is used to aggressively remove excess haematoxylin Stain procedure continues on with counterstain

B. PR0GRESSIVE - No differentiator Haematoxylin stain is applied Tissue is stained with haematoxylin only to a point Traditionally no differentiator is used to removeexcess haematoxylinStain procedure continues on with counterstain

PAP Stain Troubleshoot

Causes of inconsistent staining

Varying thickness of material on slide

Type of fixative used

Inadequate filtering of stain solutions

Age of staining solution Degree of usage of staining solutions

Use of chlorinated tap water

pH of water can effect nuclear staining

Temperature of water and reagents

Insufficient rinsing after acid

Speed of dipping slides in reagents - agitation

NB Improper draining of slides during staining.

PAP Stain Troubleshoot

PAP Stain Troubleshoot

References John D. Bancroft, Christopher Layton and

S.Kim Suvarna, (2013), Bancroft’s Theory and Practice of Histological Techniques, 7thEdition, Elsevier, China

3.J.A.Kiernan,(2015)Histological and Histochemical Methods, 5th Edition, Scion, UK

Pathcare Academy Notes