2017講座 tips for ihc and icc - antibodies, proteins, … source advantages disadvantages...

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免疫染色的完美呈現Tips on Immunohistochemistry & immunocytochemistry

Chieh-Huei Wang (王捷暉), Ph.D.

GeneTex International Corp.

β-catenin antibody GAD65 antibody

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Immunostaining (免疫染色)

H3 pSer10 (GTX128116)

HIF-α (GTX127309)

Immunocytochemistry

Ki-67 (GTX16667)

Fascin (GTX117805)

Histone H3 (GTX122148)

beta Catenin (GTX101435) BAK (GTX100063)

CK17 (GTX103765)

Immunohistochemistry

� 用標記的特異性抗體對組織切片或細胞標本中某些成分的分佈和含量進行組織和細胞原位定性、定位或定量研究的技術稱為免疫組織化學或者免疫細胞化學。

� 具有特異性強、靈敏度高、定位準確等特點

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Immunohistochemistry (IHC)

Tissue Sample Preparation

Dewaxing & Rehydrate

Antigen retrieval

Preventing Non-Specific Staining

Counterstaining

Dehydration and mounting

Visualization with microscope

1.組織樣品製作

2.脫蠟和覆水

3.抗原修復

4. 防止非特異性結合

5.抗體反應

6.呈色反應

7.複染

8.脫水&封片

9.顯微鏡觀察

� Fixatives: 4% Paraformaldehyde

� Specimen thickness: 3–4 mm

� Volume: Tissue : Fixatives = 1:20

� Time: 6 hours at least

Fixation 固定 www.genetex.com



Antigen retrieval


Immuno- staining

Primary & Secondary antibody

Chromogen substrate

Counterstaining



To preserve tissue morphology and retain the antigenicity of

the target molecules

Estrogen receptor staining

3 Hours 8 Hours

保留組織型態與抗原

Paraformaldehyde, formaldehyde and formalin

Confused?

Formaldehyde is CH2O, the simplest aldehyde.

Formalin is the name for saturated (37%) formaldehyde solution. Thus, a

protocol calling for 10% formalin is roughly equivalent to 4% formaldehyde.

Beware though, that some solutions have methanol in them to stop

polymerization but this could have a negative effect on your sample.

Paraformaldehyde (PFA) is actually polymerized formaldehyde. "Pure",

methanol-free formaldehyde can be made by heating the solid PFA. This might

be called paraformaldehyde, but it actually isn't because it’s not the polymer

form. You can buy EM grade formaldehyde or you can make your own

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石蠟包埋 Embeding

製片 Section

脱水 Dehydration

脫水、石蠟包埋和製片

石蠟浸潤 Infiltration

5-10 µm !

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Heat slides at 60℃

Xylene 100%

Alcohol

95%

Alcohol

70%

Alcohol

50%

Alcohol

2 times

5 minutes

2 times

5 minutes

1 time

5 minutes

1 time

5 minutes

1 time

5 minutes

Dewaxing & Rehydrate 脫蠟和覆水



Antigen retrieval


Immuno- staining


Chromogen substrate

Counterstaining



GTX104577 MMP2 (Human HCC)GTX104577 MMP2 (Human HCC)

Ineffective removal of wax Effective removal of wax

二甲苯梯度乙醇 (由高到低)

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Antigen retrieval 抗原修復

1. Tissue fixation create aldehyde

cross-links between proteins

2. Antibody is unable to bind

to antigen of interest

3. Retrieval buffer unmasks antigens by breaking cross-links, allowing

the antibody to bind to the antigen of interest



Antigen retrieval


Immuno- staining


Chromogen substrate

Counterstaining



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methylene bridges

組織中部分抗原在甲醛或多聚甲醛固定過程中,發生了蛋

白之間交聯及醛基的封閉作用,從而失去抗原性; 通過抗原

修復,使得細胞內抗原決定族重新暴露,提高抗原檢測率。

Antigen retrieval 抗原修復

Heat-Induced Epitope Retrieval (HIER)

• Citrate buffer method, pH6 (GTX30936)

• EDTA method, pH8 (GTX30937)

• Antigen retrieval solution, pH10 (GTX30709)

� The pH of the HIER solutions is more important than the composition of the buffer.

� A higher pH HIER solution (pH > 6 ) may be suitable

for most antigens.

� The higher the pH, the stronger the staining, the

higher the potential damage to the morphology.

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� Temperature > 90℃

� HIER solutions

� pH6以上的抗原修復液即適用於大

多數抗體� 高pH的抗原修復液可能造成較高的

背景值或組織型態破壞

熱誘導抗原修復

Without antigen retrieval

www.genetex.comAntigen retrieval - 範例

未處理

GTX100685 SQSTM1 (Human Cervical Ca.)GTX100685 SQSTM1 (Human Cervical Ca.)

Antigen retrieval pH=6

抗原回復

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Antigen retrieval - 範例

� There is no “universal” HIER buffer that is optimal for all antigens.

� A common approach is the use of a buffer such as citrate for most antigens. The high pH

or EDTA based solution may be reserved for those antigens which may be difficult to

retrieve with citrate. 抗原修復決定於酸鹼值

GTX100685 SQSTM1 (Human Breast Ca.)GTX100685 SQSTM1 (Human Breast Ca.)


Heating Source Advantages Disadvantages

Microwave

• Inexpensive

• Easy to use

• Reaches temperature rapidly

• Uneven distribution of heat

• Aggressive boiling action

• Loss of tissue

• Buffer evaporation

Vegetable Steamer

• Inexpensive

• Easy to use

• Even distribution of heat

• Good tissue morphology

• Requires more heating time than

microwave or pressure cooker

Water Bath

• Easy to use


• Good tissue morphology

• Expensive

• Requires more heating time than

microwave or pressure cooker

Pressure Cooker


• High temperature

Higher sensitivity of IHC

Short heating time required

• Expensive

• Artifacts and damaged tissue due

to high temperature

� Time / Heat source

Antigen retrieval

Proteolytic-Induced Epitope Retrieval (PIER)

• Trypsin method (GTX30934)

• Pepsin method (GTX30935)

• Pronase method (GTX73181)

• Proteolytic antigen retrieval (GTX28212)

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Room Temperature Epitope Retrieval (RTER)

• Hydrochloric Acid Method (pH 1)

• Formic Acid Method (pH 2)

� BrdU and beta amyloid

� beta amyloid

酶誘導抗原修復

特殊染色抗原修復



Antigen retrieval


Immuno- staining


Chromogen substrate

Counterstaining



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� HRP � H2O2

Endogenous Peroxidase Blocking Kit (GTX30967)

� AP� Levamisole

Endogenous Alkaline phosphatase Blocking Kit (GTX30968)

滅活內源性酶

免疫酶標技術是目前最常用的技術。先以酶標記的抗體

與組織或細胞作用，然後加入酶的底物，生成有色的不

溶性產物，通過光鏡對細胞表面和細胞內的各種抗原成分進行定位研究。

Endogenous enzymes found in a variety of cells and tissue types.

Peroxidase Alkaline Phosphatase

Red Blood Cells

Placenta

Intestine — situated between cellular

components of mucosa

Granulocytes Proximal tubules of kidney

Eosinophils Osteoblast in bone

HepatocytesArterial & capillary endothelial cell

surfaces

Muscle Stromal reticulum cells

Kidney Neutrophils

MonocytesFollicle and mantle zones in most

lymphoid tissue

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滅活內源性酶 – 範例

Secondary Ab

only

Without H2O2 With H2O2

Primary &

Secondary Ab



Antigen retrieval


Immuno- staining


Chromogen substrate

Counterstaining



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� 玻片上的特殊表面處理(如Poly-D-Lysine)，雖可以增

加組織貼附，但也容易導致非專一性蛋白結合。

� 免疫細胞中的 FCγ receptor 與 IgG 結可能造成高背景。

� Heat-inactivated normal serum

� Bovine serum albumin (BSA)

Protein Blocking Reagent (animal serum free) GTX30963

Blocking Solution with Bovine Serum (ready to use) GTX30970

Blocking Solution with Chicken Serum (ready to use) GTX30971

Blocking Solution with Donkey Serum (ready to use) GTX30972

Blocking Solution with Goat Serum (ready to use) GTX30973

Blocking Solution with Horse Serum (ready to use) GTX30974

Blocking Solution with Rabbit Serum (ready to use) GTX30975

Blocking Solution with Fish Serum (ready to use) GTX85478

Blocking

� 封閉血清一般是和二抗同一來源。血清中動物自身的抗體，預先能和組織中有交叉反應的位點發生結合。

� 也可以用小牛血清、BSA、羊血清等，但不能與一抗

來源一致。

Choose suitable 1º Ab



Antigen retrieval


Immuno- staining


Chromogen substrate

Counterstaining



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� 搜尋

� 要做什麼實驗 ?

� 要辨認的物種 ?

抗體

物種

mouse anti human p53 antibody

一級抗體選擇

Choose suitable 1º Ab 一級抗體選擇

PD-L1 antibody (GTX104763)

1:100 1:400 1:1000

稀釋倍數的影響–範例

GeneTexPD-L1 (GTX104763)

1:1000

CSTPD-L1 (#13684)

1:50

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Host Species of Primary Antibody

Class and Subclass of Primary Antibody

� The secondary antibody has to be directed against the isotype of the primary antibody.

Choose compatible 2o Ab

� The secondary antibody is raised against the host species used to generate the

primary antibody

二級抗體選擇

GTX213110-01

Goat anti Rabbit IgG antibody (HRP)

一.直接法：

標記物(熒光素和酶等)直接標記在特異性一抗上，

標記抗體和抗原直接結合

二. 間接法:

標記物(熒光素和酶等)標記在二抗上，通過一抗與抗原結合

染色方法www.genetex.com

GFAP antibody (GTX108711) Glutamine synthetase

antibody (GTX630654)

Ac-Tubulin antibody

(GTX16292)

1. Peroxidase anti-peroxidase Method

2. Streptavidin-Peroxidase Methed

3. Avidin-Biotin-peroxidase Complex Method

三. 訊號放大:PAP法

SP法

ABC法

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Endogenous biotin or lectins

Biotin Blocker

Avidin / Biotin Blocking Kit (GTX30966) StreptAvidin / Biotin Blocking Kit (GTX30965)

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內源性生物素與凝集素



Antigen retrieval


Immuno- staining


Chromogen substrate

Counterstaining



www.genetex.comChromogenic IHC Staining呈色

Β-Catenin GTX101435 ABCC4-MRP4 GTX89659

Enzyme Chromogen / Substrate Color

Horseradish

peroxidase

(HRP)

AEC GTX30938 Red

DAB GTX30939 Brown

TMB-IHC GTX30947 Blue

Alkaline

phosphatase

(AP)

BCIP/NBT GTX30943 Blue ~ Purple

Fast Red GTX30942 Red



Antigen retrieval


Immuno- staining


Chromogen substrate

Counterstaining



www.genetex.comCounterstaining 複染

• Hematoxylin (GTX73341)

• Nuclear Fast Red (GTX73309)

• Light Green (GTX73306)

• Methylene Blue (GTX73307)

目的是形成細胞輪廓，從而更好地對目標蛋白進行定位，經常用蘇木素複染（胞核染料）。

Xylene100%

Alcohol

95%

Alcohol

75%

Alcohol

Mounting封片

ImmunoHistoMount™ (GTX30922)

Cytoplasm / HPRT

(GTX113466)

Nucleus / Histone H3

(GTX129774)

Membrane PRRG2

(GTX114668)

1. 染色強度

通過在光學顯微鏡下對組織切片整體染色程度進行評分, 通常為0~3分, 分為陰

性著色(0分,－); 淡褐色(1分,+); 淺褐色(2分,++) ; 深褐色(3分,+++) 。

2. 陽性著色細胞比例

在固定倍率光鏡下，隨機選擇不重疊的3~10個視野，計數陽性著色細胞比例。

通常以百分比表示。

3. 評分法

通過在光學顯微鏡下對組織切片分別按染色程度（0~3分為陰性著色、淡黃色、

淺褐色、深褐色）、陽性範圍進行評分（1~4分為0~25%、26~50% 、51~75%、

76~100%），最終可以分數相乘或相加。

4. 灰密度分析法

在同條件下用image j進行灰密度分析，然後進行統計分析。

IHC 結果如何分析？

GTX100519GTX115549GTX101507

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Frozen Section冷陳切片

冷陳切片優點

能夠較完好地保存細胞膜表面和細胞內多種酶活性以及抗原的免疫活性

缺點

細胞內易形成冰晶而破壞細胞結構，可能會使抗原彌散

防止組織中冰晶形成的方法：

(1) 速凍

使組織溫度驟降, 減少冰晶的形成。

(2)應用蔗糖溶液

將組織置於20％～30％蔗糖溶液l～3天, 利用高滲吸收組織中水分, 減少組織含

水量, 可防止或減少冰晶的形成。

Immunofluorescence Frozen sections of mouse brain.

Vimentin antibody (GTX112661) N-cadherin antibody (GTX127345) Tuj1 antibody (GTX631836)

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保存期限?!

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Immunocytochemistry (ICC)

Label of proliferation cells

Histone H3 pSer10 (GTX128116)

Tubulin (GTX11304)

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Procedure of ICC

Cell

Preparation

Fix &

PermeableBlocking

Antibody

Incubation

Nuclear

stainingMounting

細胞製備固定& 細胞通透血清封閉抗體反應複染封片

细胞可直接培养在盖玻片上、培养瓶内或培养板上，也可将一定量的细胞收集离心制成涂片。

Cell preparation 細胞製備

� 注意细胞状态及贴盘状况

� 避免细胞过度生长重迭

Fix and Fixatives 固定和固定劑

� 活細胞標本製備好後應立即固定, 以防止細胞自溶, 保持結構, 保存細胞內的抗原性

使抗原不失活，不發生彌散和保證免疫組化定位準確。

� 標本固定後必須徹底清洗、除去多餘的固定液。

Fixatives

固定液

Cross-linking

reagents Formalin

� Preserve cell structure

� Reduce the antigenicity of some cell components

Organic solvents

Acetone � Very quick, fixes and permeabilizes at the same

� Remove lipophilic entities such as membrane lipids,

or fatMethanol

Ethanol

目前尚未發現一種通用的固定劑, 對各種不同的抗原都有滿意的固定效果。

必要時,可用多種固定液作比較, 從中挑選出對某種抗原效果最好的固定液。

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Cell

Preparation

Fix &

PermeableBlocking

Antibody

Incubation

Nuclear

stainingMounting

Permeable 细胞通透

� 目的是使抗體能夠充分地進入胞內進行結合反應。

� Triton X-100可以溶解細胞膜、細胞核膜、細胞器膜上的脂質而使抗體及大分子結

構的物質進入胞漿和胞核內，故在細胞免疫組化時尤為推薦使用，這樣抗體就能

順利進入胞內與相應抗原結合。

Cell

Preparation

Fix &

PermeableBlocking

Antibody

Incubation

Nuclear

stainingMounting

PBS blocking solution with BSA (GTX48881)

TBS blocking solution with BSA (GTX48882)

Blocking solution with Donkey Serum (GTX30972)

Blocking solution with Goat Serum (GTX30973)

Blocking solution with Horse Serum (GTX30974)

Blocking solution with Rabbit Serum (GTX30975)

Blocking 血清封閉� 為防止內源性非特異性蛋白抗原的結合，需要在

一抗孵育前先用血清（與二抗來源一致）封閉，減弱背景著色。

� 血清封閉的時間是可以調整的，一般30-60min。

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GFAP antibody α Tubulin antibody

www.genetex.comMulticolor Staining

* Different species

Mouse Ab + Rabbit Ab

* Different Conjugations

Ab-FITC + Ab-Cy3

* Different isotypes

IgG1 Ab + IgG2a Ab

Lamin A/C (GTX101127)

Tubulin (GTX11304)

Actin (GTX100315)

Tubulin (GTX11304)

Cell

Preparation

Fix & Permeable

BlockingAntibody

Incubation Nuclear staining

Mounting

一抗反應溫度� 4度、室温、37度

一抗反應時間�一般37度、4度過夜

� 建議一抗反應在4度較佳，時間最好超過16~24h。

二抗反應條件：室温或37度30min-1h

Cell

Preparation

Fix & Permeable

BlockingAntibody

Incubation Nuclear staining

Mounting

GTX30920

• DAPI (GTX16206)

• Hoechst

• PI (GTX14083)Fluoroshield™ with DAPI

複染 &封片

複染 &封片二合一

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Slide 36- Confidential -GeneTex ©

Coverslip

Number Thickness (mm)

#0 0.08 – 0.13

#1 0.13 – 0.16

#1.5 0.16 – 0.19

#2 0.19 – 0.25

#3 0.25 – 0.35

#4 0.43 – 0.64

Objective

GTX100865 Synaptophysin

Confocal microscope In-house microscope

PMID: 14578425

免疫染色常見問題解答www.genetex.com

� 確認是否忽略了應該加的某種試劑，包括一抗、二抗、三抗及底物等。

� 確認所有的試劑是否按正確的順序加入，是否反應了足夠的時間。

� 確認所用的檢測系統是否和一抗匹配。

� 檢查抗體所使用的稀釋度，有效期和保存條件。

� 檢查標本的儲存條件，最好用已知陽性的標本來同時做陽性對照。

� 檢查色原/底物溶液，最簡單的檢測方法是將一滴標記有酶的抗體加入到製備好

的底物溶液中，如果底物發生預期的顏色變化，則可排除底物的因素。

標本無染色 or 弱陽性

� 固定不全或過度固定

� 脫蠟不全

� 抗原修復不全

� 細胞通透不全，抗體未能充分進入胞內參與反應 (核蛋白特別易發生)

� 細胞通透破壞抗原(膜蛋白特別易發生)

� 組織切片本身這種抗原含量低

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免疫染色常見問題解答

� 抗體的使用濃度是否過高, 孵育時間是否恰當。

� 是否有效地去除了內源性酶及生物素。

� 是否選擇使用了正確的封閉血清。

� 清洗是否充分,清洗液是否保存適當。

� DBA的使用是否正確。

� 標本染色過程中乾涸會造成邊緣部的非特異性染色。

� 檢查二抗與標本的內源性組織蛋白是否有交叉反應。

� 過度的放大。

非特異性染色

Substrate

• Endogenous enzyme may be developing the substrate.

Blocking

ABC or SP

Substrate

• ABC reagent may be binding to tissues.

• Certain grades of BSA may contain contaminants (bovine IgG, lipids, etc.)

that can contribute to background staining.

Blocking

2oC Antibody

ABC or SP

Substrate

• Secondary antibody may bind nonspecifically to tissue components.

• Cross-reactivity may occur between secondary antibody and endogenous

immunoglobulins or other tissue proteins.

• The wrong species of blocking serum was used.

Blocking

1oC Antibody

2oC Antibody

ABC or SP

Substrate

• Too much primary antibody has been used.

• The primary antibody may cross-react with other tissue epitopes or bind

nonspecifically.

• If the section shows small, amorphous, punctate staining, the primary

antibody may have some denatured precipitated immunoglobulin.

問題排除測試方法www.genetex.com

THANK YOU

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USA

Toll-free : 1.877.GeneTex(1.877.436.3839)

Fax : 949.309.2888

Address : 2456 Alton Parkway, Irvine, CA 92606

E-mail : [email protected]

International

Tel : 886.3.6208988

Fax : 886.3.6209098

Address : 6F-2, No.89, Dongmei Rd., East Dist., Hsinchu City 300, Taiwan (R.O.C.)

E-mail : [email protected]

1. 外鍋放水(量要足夠使電鍋在40分鐘內都不會煮乾)

染色專用缸(勿用玻璃會爆炸)

Retrieval buffer(量要能蓋過tissue)

2. 於 retrieval 步驟前10分鐘先預熱

3. 待外鍋沸騰時, 將sample放入染色

專用缸

4. 蓋上鍋蓋計時30分鐘

Sample

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2017講座 tips for ihc and icc - antibodies, proteins, … source advantages disadvantages...

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