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Immunohistochemistry

(IHC)

 

Immunohistochemistry (IHC) is a method for detecting  target protein in tissue sections by exploiting the principle of antibodies binding specifically to antigens in biological tissue. The antibody-antigen binding can be visualized in different ways. Enzymes, such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP), are commonly used to catalyze a color-producing reaction.

IHC is widely used in experimental and clinical research because this technique makes it possible to visualize the distribution and localization of specific cellular components within cells and in proper tissue context. There are numerous IHC methods that can be used to localize antigens. The method selected should include consideration of parameters such as the specimen types and assay sensitivity.

 

 

I. Sample Preparation

A. Paraffin section

1. Tissue collection and fixation

Collect tissue from different specimens, then cleaning with normal saline. Use appropriate fixation to fix it.

2. Dehydration

The common dehydrate reagent is gradient alcohol. Please dehydrate from low concentration to high concentration, for skip one step can cause tissue shrinkage and deformation. The purpose of dehydration is to harden the tissue, facilitate to soak oil or transparent agent, and provide conditions for melting paraffin into the tissue.

3. Transparency

Transparency is a necessary step before cutting the specimen. Since anhydrous ethanol and paraffin are not mutually soluble, we need to replace anhydrous ethanol with a transparent agent, such as mineral oil or vegetable oil. The commonly transparent agent includes xylene, benzene and methylbenzene.

4. Embedding.

After transparentizing, the tissue can be immersed in molten paraffin wax so that it adsorbs the wax-substituting transparent agent. Based upon the melting point of wax, immersion should be performed at 54-64℃.

Embedding is a process of treating the tissue in a paraffin box so that the paraffin wax cools down and solidifies. The treatment conditions (using ethanol and xylene as an example) are shown in the table below. After cooling is completed, the tissue will be ready for sectioning and suitable for storage.

5. Shaping and slicing

The paraffin block was fixed and trimmed before slicing. The tissue was sliced by paraffin slicing machine and adhered to the slide glass. Roast at 60℃ for 2h in the oven.

B. Frozen section

1. Tissue Collection and fixation

Collect tissue from fresh specimens, then cleaning with normal saline. Use appropriate fixation to fix it.

2. Freezing and slicing

The common freezing methods are using CO2and dry ice.The tissue was cut by freezing slicing machine and adhered to the pretreated slide glass. Carry on subsequent experiment or preserve at -80℃ after drying at room temperature.

P1110

Paraformaldehyde,4%

Applied in light microscope, Immunochemical

P1111

Paraformaldehyde,1%

Applied in electron microscopy to observation ultrastructure of tissue

P1112

Paraformaldehyde,8%

P1116

Paraformaldehyde-glutaraldehyde Fixative Solution(2%/2.5%)

P1127

Glutaraldehyde,4% (EM Grade)

 

YA0010

Paraffin wax, 48-50℃

YA0011

Paraffin wax, 58-60℃

 

YA0012

Paraffin wax, 56-58℃

 

YA0014

Paraffin wax, 60-62℃

 

YA0013

Paraffin wax, 54-56℃

 

YA0015

Paraffin wax, 50-52℃

 

YA0031

Dewaxing Solution

 

P8120

Poly-L-lysine hydrobromide mol wt 30,000-70,000

 

P8130

Poly-L-lysine hydrobromide mol wt 70,000-150,000

 

P8140

Poly-L-lysine hydrobromide mol wt 150,000-300,000

 

P2100

Poly-L-lysine Solution,10×

 

Note: Paraffin section should be preserve at room temperature or 4℃. Frozen section should be preserve at -80℃.

II. Deparaffinating and hydration

Paraffin section should be deparaffinating in xylene, and then hydration in gradient ethanol solution.  

YA0310

Immunohistochemical pen

Note:

1. Incomplete deparaffinating can affect the result; suggest deparaffinating in oven before using xylene for dewaxing.

2. Immunohistochemical pen can outline the tissue after hydration, saving reagent dosage.

III. Antigen retrieval

Formaldehyde fixation usually generates methylene bridges which cross-link proteins and therefore mask the epitope of interest. It is essential to unmask the antigen epitopes in order to allow the antibodies to bind, either by heat or enzymatic digestion.

C1031

Sodium Citrate Antigen Retrieval Solution,1×

Heat Induced Epitope Retrieval

C1032

Sodium Citrate Antigen Retrieval Solution,50×

C1033

EDTA Antigen Retrieval Solution,1×

C1034

EDTA Antigen Retrieval Solution,50×

C1035

Antigen Retrieval Solution,1×(for Frozen Sections)

C1036

Antigen Retrieval Solution,1×(for IHC)

T8150

Trypsin 1: 250

Applied in intracellular antigen

X1020

Trypsin Digestion solution,0.1%

P9460

Proteinase K

P8360

PronaseE from Streptomyces griseus

P8390

Pepsin 1: 3000

Applied in intercellular antigen

P8160

Pepsin 1: 10000 from porcine gastric mucosa

Note: The selection of antigenic repair solution depends on the type of slice, the location and properties of antigenic cluster; Excessive digestion will damage the tissue, so you should choose proper digestion time and then wash sufficiently.

IV. Membrane Permeabilization

Note: If antigen epitopes are distributed on the cell surface, the step can be omitted.)

Triton X-100 is frequently used in permeabilization, the treatment helps to expose antigen epitomes, so that antibodies can more easily enter cells, thus ensuring the correctness of dyeing results.

T8200

Triton X-100

Working solution range: 0.1%-1%

V. Inactivation and blocking endogenous biotin

There is a certain amount of endogenous enzyme and endogenous biotin in organism tissue. Such as blood-rich tissue contain a large number of active peroxidase, liver, spleen, kidney, brain and embryonic tissue contain rich biotin. Both the endogenous enzyme and biotin can interference experiment results and cause a false positive.

(1) Inactivation of Endogenous peroxidase

For paraffin embedded section: incubate in 3% H2O2 for 10 min.

For frozen section or cell section: incubate in solution composed of methanol and 3% H2O2 (v/v: 4: 1) for 30 min, as methanol passivates all the enzymes

(2) Inactivation of endogenous alkaline phosphatase:

Incubate the sample section in 24mg/mL Levamisole at pH 7.6~8.2, it can inhibit mostly endogenous AP, but cannot inhibit the AP activation of endogenous intestine tissue. The Inactivation of acid phosphates can inhibit by 0.05 mol/L tartaric acid.

(3) Blocking endogenous biotin:

Incubate the sample section in 0.01% avidin solution at room temperature for 10-20 minutes, saturate the binding sites to eliminate endogenous biotin activity.

VI. Blocking

Residual sites on the tissue section may bind to secondary antibody and produce follow-up false positive results. Therefore, BSA, skim milk, serum from the same species as the secondary antibody is commonly used for blocking.

SW3015

5% BSA Blocking Buffer

 

D8340

Skim Milk

 

SL042

Normal Equine Serum

Usually the serum comes from unimmunized animals

SL038

Normal Goat Serum

SL034

Normal Rabbit Serum

SL039

Normal Sheep Serum

SL035

Normal Chicken Serum

SL050

Normal Donkey Serum

Note: When BSA or skimmed milk leads to a high background, please use the unimmunized serum instead.

VII. Specific immunological reaction

The key of IHC is to have a good quality primary antibody. We also provide you various dilution buffer and antibody.

A1810

Primary Antibody Dilution Buffer

A1800

Antibody Dilution Buffer

A1820

HRP-conjugated Antibody Dilution Buffer

A1830

AP-conjugated Antibody Dilution Buffer

A1840

Fluorescent Antibody Dilution Buffer

SE131

Goat anti-Mouse IgG/HRP

SE134

Goat anti-Rabbit IgG/HRP

SE240

Rabbit anti-Guinea pig IgG/HRP

SE265

Rabbit anti-Avidin/HRP

SF134

Goat anti-Rabbit IgG/FITC

SF131

Goat anti-Mouse IgG/FITC

SF240

Rabbit anti-Guinea pig IgG/FITC

Note: The dilution ratio of primary antibody need to refer the instruction, suggest store at -20℃ after subpackage.

VIII. Chromogens

The selection of secondary antibody depends on the source of the primary antibody. For example: the primary antibody is rabbit anti-human, and then the second antibody should choose mouse or goat anti rabbit. The common chromogenic methods are Enzyme colorimetry and fluorescent detection.

SABC

SA0011

SABC(Mouse IgG)-POD Kit

Suitable for anti-mouse IgG IHC detection.

SA0012

SABC(Mouse IgG)-AP Kit

SA0013

SABC(Mouse IgG)-FITC Kit

SA0015

SABC(Mouse IgG)- FITC(POD) Kit

SA0021

SABC(Rabbit IgG)-POD Kit

Suitable for anti-rabbit IgG IHC detection.

SA0022

SABC(Rabbit IgG)-AP Kit

SA0023

SABC(Rabbit IgG)-FITC Kit

SA0025

SABC(Rabbit IgG)-FITC(POD) Kit

SA0031

SABC(Goat IgG)-POD Kit

Suitable for anti-goat IgG IHC detection.

SA0032

SABC(Goat IgG)-AP Kit

SA0033

SABC(Goat IgG)-FITC Kit

SA0035

SABC(Goat IgG)-FITC(POD) Kit

SA0041

SABC(Mouse/Rabbit IgG)-POD Kit

Suitable for anti-mouse or rabbit IgG IHC detection..

SA0042

SABC(Mouse/Rabbit IgG)-AP Kit

SA0043

SABC(Mouse/Rabbit IgG)-FITC Kit

SP

SP0011

SP(Mouse IgG)-POD Kit

Suitable for anti-mouse IgG IHC detection.

SP0021

SP(Rabbit IgG)-POD Kit

Suitable for anti-rabbit IgG IHC detection.

SP0031

SP(Goat IgG)-POD Kit

Suitable for anti-goat IgG IHC detection.

SP0041

SP(Mouse/ Rabbit IgG)POD Kit

Suitable for anti-mouse or rabbit IgG IHC detection.

Note: The chromogenic reaction should be proceed under microscope in real-time and stop the reaction immediately after color rendering

Counterstain

In general, after staining the target antigen by IHC, a secondary stain is usually applied to provide contrast that helps the primary stain more distinct. The selection of the counterstain depend on the IHC staining. The two dye must be quite different, and the stain time varied among the different dye.

G1080

Mayer' Hematoxylin solution (For IHC)

the nucleus are stained blue

G1320

Nuclear Fast Red solution, 0.1%

the nucleus are stained red

G1321

Nuclear Fast Red solution, 0.2%

G1651

Methyl green,0.5%

the nucleus are stained green

G1650

Methyl green,0.1%

IX. Mounting Medium

G8590

Neutral balsam

S2150

Glycerol Gelatin aqueous slide mounting medium

S2100

Mounting Medium, antifading

S2110

Mounting Medium, antifading (with DAPI)

Note: Coverslip press slowly from one side to the other side during sealing avoids bubbles.

Frequently Asked Questions:

1. Neither the control nor the sample was stained.

1) Confirm whether the operation steps are omitted or reversed, and the preservation conditions and shelf life especially the antibodies.

2) According to the label, reconfirm whether the source of primary and secondary antibodies match, and whether the dilution of antibody is correct.

3) Examine the quality of sample, suggest use known positive sample as reference.

4) Examine substrate reagent, the simple method is adding an enzyme labeled antibody to the prepared substrate solution. If there is a color change, the substrate factor can be excluded.

5) Examine the wash solution and dilution buffer, the PH of solution is important. For example, sodium azide inhibits peroxidase activity, so the solution used in the experiment cannot contain sodium azide.

6) Examine whether the selection of counterstain and mounting medium are matching.

2. Weak positive

If negative control had no reaction and positive control and sample present weak positive.

1) Examine the fixation and antigen retrieval method whether fitted with target antigen.

2) Whether the antibody dilution is too high or the incubation time is enough.

3) If there is still some washing liquid left on the slice, then it is equivalent to further dilution of the antibody artificially.

4) The slice should be placed horizontally, otherwise the antibody will loss. If negative control had no reaction, the positive control react well, while the sample is weakly positive, it may be because the positive control and sample are not the same tissue, or fixed in different ways etc.

3. High background.

1) Whether the endogenous enzyme is inactivation and endogenous biotin has been blocked.

2) Whether the selection of blocking serum is correct.

3) Whether the specificity of the primary antibody is good, and whether the concentration of the primary antibody and secondary antibody is too high.

4) After the incubation of the primary and secondary antibody, it is needed to clean thoroughly..

5) Whether the use of color solution is strictly in accordance with the instructions

6) Whether the specimen has dried up during the staining process, which results in nonspecific staining on the edge.

7) Examine whether the secondary antibody has cross reaction with endogenous tissue protein.


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