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.

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.  

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.

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.

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.

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.

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.

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.

IX. Mounting Medium

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.