IHC Full Process Operation Video

Paraffin Section IHC-P Experimental Operation Procedure

1. Dewaxing to Water

Before staining paraffin sections, dewaxing treatment is necessary to ensure successful staining of the sections.

1) Xylene I for 10 minutes;

2) Xylene II for 10 minutes;

3) Xylene III for 10 minutes;

4) Absolute ethanol I for 3 minutes;

5) Absolute ethanol II for 3 minutes;

6) 95% ethanol for 3 minutes;

7) Running water rinse for 3 minutes.

2. Antigen Retrieval

Commonly used antigen retrieval methods include high-pressure retrieval, water boiling retrieval, and enzyme retrieval; commonly used retrieval solutions include citric acid solution (pH 6.0), EDTA solution (pH 9.0), Tris-EDTA (pH 9.0), trypsin, pepsin, etc.

1) Water boiling retrieval: Add an appropriate amount of 1×Tris-EDTA (pH9.0)/citric acid (PH6.0) buffer to a high-temperature container, heat it with an induction cooker until it boils for 3-5 minutes to ensure no bubbles under low heat; place the tissue sections in the container, adjust the induction cooker to low power to maintain the temperature at 90-95°C for 20 minutes, and cover with a lid. After cooling at room temperature for 10 minutes, rinse with running tap water to cool to room temperature. (For tissues that easily detach, such as bone, 60°C overnight repair can be used.)

2) High pressure retrieval: Add an appropriate amount of 1×citric acid (pH6.0) buffer to a pressure cooker, heat to boiling without a lid, place the tissue sections in the pressure cooker, add the valve, heat until uniform steam is emitted, maintain for 2 minutes, cool at room temperature for 10 minutes, then open the pressure cooker lid after the pressure valve drops, and rinse with running water to cool to room temperature.

Note: This method is suitable for antigen retrieval of difficult-to-detect or nuclear antigens.

3) Enzyme retrieval: After slightly drying the sections, use filter paper to wipe dry the water around the tissue, draw a circle around the tissue with a histochemical pen (to prevent antibody runoff, note that the histochemical pen tip should not get wet, and the circle should be appropriately sized, about 0.5cm from the tissue), add trypsin working solution within the circle to cover the tissue, place the sections flat in a wet box and incubate at room temperature for 20 minutes, rinse with distilled water 3 times, 3 minutes each time.

Note: Reason for antigen retrieval: During formaldehyde or paraformaldehyde fixation, some antigens in the tissue undergo protein cross-linking and aldehyde group blocking, resulting in loss of antigen activity. Through antigen retrieval, intracellular antigenic determinants can be re-exposed, improving the success rate of antigen detection.

3. Immunohistochemical Staining

1) Wash the sections with pure water three times, 5 minutes each time.

2) Blocking: Place the sections in 3% hydrogen peroxide aqueous solution and incubate for 10 minutes.

Note: Reason for blocking: Granulocytes, monocytes, and red blood cells in tissues contain endogenous peroxidase, which, like horseradish peroxidase, can react with chromogenic agents DAB and AEC to cause false positives. The method to eliminate endogenous peroxidase is to block with hydrogen peroxide solution.

3) Wash the sections with pure water twice, 5 minutes each time.

4) After slightly drying the sections, use filter paper to wipe dry the water around the tissue sections, draw a circle around the tissue with a histochemical pen, add 10% negative goat serum prepared with 1% BSA within the circle to cover the tissue, and block at room temperature for 1 hour.

5) Remove the blocking solution, then add primary antibody diluted with the recommended antibody diluent to each section (usually 100μL/tissue), and incubate at room temperature in a wet box for 1 hour.

6) Remove the antibody solution, rinse with 1×TBST 3 times, 5 minutes each time.

7) After patting the sections dry, add goat anti-rabbit or mouse secondary antibody (HA1119 or HA1120) within the circle to cover the tissue, and incubate at room temperature in a wet box for 20 minutes.

*HA1119 (HRP Conjugated Goat anti-Rabbit IgG UltraPolymer Goat Polyclonal Antibody)

*HA1120 (HRP Conjugated Goat anti-Mouse IgG UltraPolymer Goat Polyclonal Antibody)

8) Rinse with 1×TBST 3 times, 5 minutes each time.

9) DAB color development: After patting the sections dry, add freshly prepared DAB chromogenic solution within the circle, control the color development time under a microscope, positive is brownish-yellow, observe until positive signals appear or develop for 5 minutes, rinse the slides with a wash bottle filled with tap water or a dedicated DAB beaker to stop color development, and rinse with running tap water for 5-10 minutes.

Note: DAB reagent has potential mutagenic effects, double gloves should be worn for protection during preparation and color development, and dedicated washing beakers should be used after DAB color development, not mixed with other beakers.

4. Counterstaining and Dehydration

1) Hematoxylin for 10 minutes;

2) Running water rinse for 3 minutes;

3) Blueing agent for 1 minute;

4) Running water rinse for 3 minutes;

5) 95% ethanol I for 1 minute;

6) 95% ethanol II for 1 minute;

7) 95% ethanol III for 1 minute;

8) Absolute ethanol I for 1 minute;

9) Absolute ethanol II for 1 minute;

10) Xylene I for 1 minute;

11) Xylene II for 1 minute;

12) Oven at 65°C for 2 minutes.

*Reagents need to be replaced regularly, and blueing solution can be replaced in advance if discolored.

5. Mounting

Mount with neutral gum.

Note: During the entire operation, the sections should be kept in a moist environment to avoid drying, otherwise non-specific staining is likely to occur.

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