IHC Protocol

<Formalin fixed paraffin embedding section>

PREPARATION OF EQUIPMENT AND REAGENT

• Equipment

1. A stainless steel pressure cooker or a microwave oven or an electric heater
2. A water bath pot

• Reagent

1. APES or POLY-L-LYSINE
2. Dimethylbenzene, absolute ethyl and ethanol
3. PBS buffer (pH7.2-7.4)
4. Endogenous peroxidase blockin agent
5. 0.01 mole / L citrate buffer (pH6.0) or 0.5 mole / L EDTA buffer (pH8.0)
6. Enzymatic digestion solution: 0.1% trypsinase or 0.4% pepsase
7. Normal goat serum or 5% BSA
8. DAB, haematoxylin and neutral balsam mounting medium
9. TBS or PBS (pH9.0-9.5) for fluorescence microscope, (pH7.0-7.4) for optical microscope

ASSAY PROCEDURE

1. Cover the entire surface of a clean microslide with APES or POLY-L-LYSINE. Incubate for 1 minute then rinse the microslide with water. Mount a tissue section (~5μm thick) with the treated microslide and bake in an oven at 60˚C for 30-60 minutes to ensure strong adhesion of the tissue section.
2. Dewax the tissue section.

1. Dewax the tissue section in dimethylbenzene for 10 minutes, then, immerse the tissue section for another 10 minutes in the fresh dimethylbenzene.
2. Immerse the tissue section in absolute ethyl alcohol for 5 minutes.
3. Immerse the tissue section in 95% ethanol for 5 minutes.
4. Immerse the tissue section in 70% ethanol for 5 minutes.
5. Rinse the tissue section with water.

3. Wash the tissue section with PBS buffer (pH 7.2~7.4) for 2~3 times, 5 minutes for each.
4. Incubate the tissue section for 5~10 minutes with endogenous peroxidase blocking agent to quench the endogenous peroxidase activity.
5. Wash the tissue section with PBS buffer (pH 7.2~7.4) for 2~3 times, 5 minutes for each.
6. Antigen retrieval.

>> Antigen retrieval by heat (For different antigens, here are three methods to heat repair ).

1. By high pressure Add 0.01M citrate buffer (pH6.0) or 0.5 M EDTA (pH8.0) into the boiling water of the stainless steel pressure cooker. Soak the tissue section in the above buffer for 5 minutes, then, cover the cooker and heat for 10 minutes, and stop heating. This method is suitable for nuclear or hard detection antigens.
2. By boiling Heat the 0.01M citrate buffer (pH6.0) with an electric heater or a water bath pot  to about 95˚C, put the tissue section in and heat for 10~15 minutes.
3. By microwave Soak the tissue section in 0.01M citrate buffer (pH6.0), and heat to the boiling point with a microwave oven, then stop heating. Repeat this heating process 1~2 times with a 5~10-minute interval. 

>> Antigen retrieval by enzymatic digestion

0.1% trypsinase or 0.4% pepsase is usually used as enzymatic digestion solution. Protocol: incubate the tissue section in 0.1% trypsinase at 37˚C for 5~30 minutes; or in 0.4% pepsase at 37˚C for 30 minutes.

7. Wash the tissue section with PBS buffer (pH 7.2~7.4) for 2~3 times, 5 minutes for each.
8. Add normal goat serum (blocking solution) or 5% BSA to the tissue section and incubate at room temperature for 20 minutes. Discard the blocking solution, but do not wash the tissue section.
9. Add 50µl of properly diluted primary antibody to the tissue section and incubate at 37˚C for about 1 hour or 20˚C for about 2 hours or at 4˚C overnight (If 4˚C overnight, equilibrate the tissue section at 37˚C for 45 minutes). Wash the tissue section with PBS (pH 7.2~7.4) 3 times for 5 minutes each. (The primary antibody concentration, incubation time and temperature directly affect the staining efficiency and background intensity. If the positive staining is too weak, the concentration of the primary antibody and the incubation time can be increased; if the background is too high, the primary antibody concentration and the incubation time can be decreased. )
10. Add 40~50µl of properly diluted secondary antibody to the tissue section and incubate at 37˚C for 1 hour (0.05% Tween-20 can be added into the secondary antibody). Wash the tissue section with PBS (pH 7.2~7.4) 3 times for 5 minutes each.
11. Stain the tissue section with DAB chromogenic kit for 5~10 minutes, observe the staining effect under a microscope. Wash the tissue section with PBS (pH 7.2~7.4) or distilled water for 10 minutes.
12. Slightly counterstain the tissue section with haematoxylin for 2 minutes, and wash with distilled water for 10~15 minutes to clean the haematoxylin. Then dry the tissue section by baking, and put on a drop of neutral balsam mounting medium, and seal the tissue section with a cover slide. The tissue section is ready for observation under a microscope.