Detailed Immunohistochemistry (IHC) Staining Protocol (For Reference Only)

The core of Immunohistochemistry (IHC) lies in the specific binding between antigens and antibodies. By labeling target antigens with chromogens (e.g., DAB), it enables the localization and expression level analysis of specific proteins in tissue sections. Key steps such as deparaffinization and antigen retrieval must be strictly controlled to ensure the reliability of results. The detailed procedures are as follows:

1. Pre-Experimental Preparation

1.1 Reagent Preparation

Basic Reagents: Xylene (for deparaffinization), gradient ethanol (100%, 95%, 85%, 75% for rehydration after deparaffinization), PBS buffer (pH 7.4 for slide washing), 3% H₂O₂ solution (for blocking endogenous peroxidase).

Core Reagents: Antigen retrieval buffer (citrate buffer pH 6.0 or EDTA buffer pH 8.0, selected based on antibodies), blocking solution (5% BSA or normal goat serum), primary antibody (diluted per manual), HRP-conjugated secondary antibody (diluted per manual), DAB chromogenic solution, hematoxylin staining solution (for nuclear counterstaining), dehydration and clearing reagents (gradient ethanol, xylene), neutral balsam (for mounting).

1.2 Equipment Preparation

Tissue sections (paraffin-embedded or frozen sections), slide rack, staining jar, humid chamber, pipette, microscope, coverslips, absorbent paper.

1.3 Section Pretreatment

Paraffin-embedded sections: Bake in a 60℃ oven for 1-2 hours (enhance section adhesion to slides and prevent detachment).

Frozen sections: Allow to stand at room temperature for 30 minutes or dry overnight at 4℃.

2. Deparaffinization and Rehydration of Paraffin-Embedded Sections (Skip for Frozen Sections)

Soak sections in the following order for 5-10 minutes per step to ensure complete deparaffinization:

Xylene Ⅰ → Xylene Ⅱ (remove paraffin);

100% Ethanol Ⅰ → 100% Ethanol Ⅱ → 95% Ethanol → 85% Ethanol → 75% Ethanol (gradual rehydration to avoid tissue shrinkage);

Soak in double-distilled water for 5 minutes (wash off ethanol for subsequent steps).

3. Blocking Endogenous Peroxidase

Immerse sections in 3% H₂O₂ solution (prepared with methanol or PBS), incubate at room temperature in the dark for 10-15 minutes. This step inhibits endogenous peroxidase activity in tissues to avoid interfering with subsequent DAB chromogenesis. After incubation, wash slides with PBS buffer 3 times for 3 minutes each.

4. Antigen Retrieval (Critical Step)

The purpose is to expose antigen epitopes masked by formalin fixation and ensure effective antibody binding. Select retrieval method according to antibody instructions:

4.1 Heat-Induced Epitope Retrieval (HIER, Most Commonly Used)

Immerse sections in antigen retrieval buffer and heat in a microwave oven or pressure cooker.

Microwave oven: Heat on medium-high power until boiling, turn off power and let stand for 5 minutes, repeat 2-3 times, then cool naturally to room temperature.

Pressure cooker: Heat until steaming, maintain for 1-2 minutes, turn off heat and allow natural pressure release, then cool to room temperature.

4.2 Enzyme Retrieval (Suitable for Partial Antigens)

Immerse sections in trypsin or pepsin solution, incubate at 37℃ for 10-30 minutes, and terminate the reaction by washing with PBS.

4.3 Post-Retrieval Washing

Wash slides with PBS buffer 3 times for 3 minutes each after retrieval.

5. Blocking and Antibody Incubation

5.1 Blocking

Blot dry water around sections with absorbent paper (avoid antibody dilution), add blocking solution to the tissue area, and incubate at room temperature for 30-60 minutes to block non-specific binding sites and reduce background staining.

5.2 Primary Antibody Incubation

Discard the blocking solution (no washing required), add specifically diluted primary antibody, place sections in a humid chamber (prevent antibody evaporation), and incubate overnight at 4℃ (recommended for full binding) or 2 hours at room temperature.

5.3 Washing and Secondary Antibody Incubation

After primary antibody incubation, wash slides with PBS buffer 3 times for 5 minutes each to thoroughly remove unbound primary antibody.

Add HRP-conjugated secondary antibody (species-matched with primary antibody) and incubate in a humid chamber at room temperature for 30-60 minutes.

After secondary antibody incubation, wash slides with PBS buffer again 3 times for 5 minutes each.

6. Chromogenesis and Counterstaining

6.1 DAB Chromogenesis

Take out sections, blot dry surrounding water, add freshly prepared DAB chromogenic solution, and monitor chromogenesis in real-time under a microscope (usually 1-5 minutes). Immediately rinse with double-distilled water to terminate chromogenesis when clear brownish-yellow signals appear in target areas with low background.

6.2 Hematoxylin Counterstaining

Immerse sections in hematoxylin staining solution, stain at room temperature for 3-5 minutes (stain cell nuclei blue for contrast with brownish-yellow antigen signals), and rinse with running water for 10 minutes (blueing to enhance nuclear clarity).

7. Dehydration, Clearing and Mounting

Soak sections in the following order for 3-5 minutes per step to complete dehydration and clearing:

75% Ethanol → 85% Ethanol → 95% Ethanol → 100% Ethanol Ⅰ → 100% Ethanol Ⅱ (gradual dehydration to prevent tissue deformation);

Xylene Ⅰ → Xylene Ⅱ (clearing to improve fusion between sections and mounting medium).

Mounting: Take out sections, blot excess xylene, add 1-2 drops of neutral balsam to the center of the tissue, and gently cover with a coverslip (avoid air bubbles). Allow to stand at room temperature for several hours or bake in a 37℃ oven for 30 minutes until the balsam solidifies.

8. Result Observation and Analysis

8.1 Observation

Examine sections under a light microscope. Positive signals appear brownish-yellow; determine if they correspond to the target antigen based on intracellular localization (nucleus, cytoplasm, or cell membrane).

8.2 Analysis

Qualitative Analysis: Determine "positive" or "negative" expression of the target antigen in tissues.

Semi-Quantitative Analysis: Score based on the proportion of positive cells and staining intensity (weak positive, moderate positive, strong positive) to quantify expression differences among different samples.