Protocol of Immunohistochemistry (IHC)

Immunohistochemistry (IHC) is used to understand the localization and distribution of target proteins in different parts of a biological tissue. IHC detects specific antigens in preserved tissue sections using an appropriate antibody labeling strategy. Samples are collected, fixed to maintain cell morphology, tissue architecture and antigenicity of target epitopes, and then sectioned. Our detailed IHC protocols cover all aspects of specimen preparation for IHC, whether you are staining paraffin-embedded, frozen or free-floating sections or whole tissue mounts.

Preparation and Chromogenic IHC Staining of Frozen Tissue Sections

Preparation and Fluorescent IHC Staining of Frozen Tissue Sections

Preparation and Chromogenic IHC Staining of Paraffin-embedded Tissue Sections

Preparation and Fluorescent IHC Staining of Paraffin-embedded Tissue Sections

Troubleshooting of Immunohistochemistry (IHC) is also available for you.

General Steps

• Slide Preparation
• Fixation
• Antigen Retrieval (Optional)
• Permeabilization
• Blocking and Immunostaining
• Multicolor Staining (Optional)
• Counter Staining
• Mounting

Slide Preparation

1. Coat coverslips with poly-L-lysine (or polyethyleneimine) for 1 h at RT.
2. Rinse coverslips well with sterile H₂O for three times. 1 h each time.
3. Allow coverslips to dry completely. Sterilize them under UV light for at least 4 h.
4. Grow cells on glass coverslips.
5. Rinse briefly in PBS.

Fixation

The cells may be fixed using one of two methods:

• Option 1: Incubating the cells in 100% methanol (chilled at -20°C) at RT for 5 min. The cells should be washed three times with ice-cold PBS.
• Option 2: Using 4% paraformaldehyde in PBS at RT for 10 min. The cells should be washed three times with ice-cold PBS.

Antigen Retrieval (Optional)

Certain antibodies work best when cells are heated in antigen retrieval buffer. Please check the product information for recommendations for each primary antibody being used.

1. Preheat the antigen retrieval buffer to 95°C.

Note: This can be achieved by heating the buffer in a cover glass staining jar which is placed in a water bath at 95°C. Suggested retrieval buffer: 100 mM Tris, 5% (w/v) urea, pH 9.5.

2. Using a small pair of broad-tipped forceps, place the coverslips carefully in the antigen retrieval buffer in the cover glass staining jar, making note of which side of the coverslips the cells are on.
3. Heat the coverslips at 95°C for 10 min.
4. Remove the coverslips from the antigen retrieval buffer. Immerse them with the side containing the cells facing up, in PBS, in the 6-well tissue culture plates.
5. Wash cells in PBS three times for 5 min.

Permeabilization

If the target protein is intracellular, it is very important to permeabilize the cells. For methanol fixed samples, permeabilization is not required.

1. Incubate the samples for 10 min with PBS containing either 0.1-0.25% Triton X-100.

Note: Triton X-100 is not appropriate for membrane-associated antigens since it destroys membranes. The optimal percentage of Triton X-100 should be determined for each protein of interest.

2. Wash cells in PBS three times for 5 min.

Blocking and Immunostaining

1. Incubate cells with 1% BSA, 22.52 mg/mL glycine in PBS-T for 30 min to block non-specific binding of the antibodies.
2. Incubate cells in the diluted primary antibody in 1% BSA in PBS-T for 1 h at RT or overnight at 4°C.
3. Decant the primary antibody solution. Wash three times in PBS. 5 min each wash.
4. Incubate cells with the secondary antibody in 1% BSA for 1 h at RT in the dark.
5. Decant the secondary antibody solution. Wash three times in PBS in the dark. 5 min each wash.

Multicolor Staining (Optional)

• Simultaneous Incubation
1. Incubate cells with blocking solution for 30 min.
2. Incubate cells with both primary antibodies in 1% BSA in PBS-T for 1 h at RT or overnight at 4°C.
3. Decant the primary antibody solution. Wash three times in PBS. 5 min each wash.
4. Incubate cells with both secondary antibodies in 1% BSA in PBS-T for 1 h at RT in the dark.
5. Decant the secondary antibody solution. Wash three times in PBS in the dark. 5 min each wash.
• Sequential Incubation
1. First blocking step: Incubate cells with the first blocking solution for 30 min at RT.
2. Incubate cells with the first primary antibody in 1% BSA in PBS-T for 1 h at RT or overnight at 4°C.
3. Decant the first primary antibody solution. Wash three times in PBS. 5 min each wash.
4. Incubate cells with first secondary antibody in 1% BSA in PBS-T for 1 h at RT in the dark.
5. Decant the first secondary antibody solution. Wash three times in PBS in the dark. 5 min each wash.
6. Second blocking step: Incubate cells with the second blocking solution for 30 min at RT in the dark.
7. Incubate cells with the second primary antibody in 1% BSA in PBS-T in the dark for 1 h at RT, or overnight at 4°C.
8. Decant the second primary antibody solution. Wash three times in PBS in the dark. 5 min each wash.
9. Incubate cells with second secondary antibody in 1% BSA in PBS-T for 1 h at RT in the dark.
10. Decant the second secondary antibody solution. Wash three times in PBS in the dark. 5 min each wash.

Counter Staining

1. Incubate cells on 0.1-1 μg/mL Hoechst or DAPI (DNA stain) for 1 min.
2. Rinse with PBS.

Mounting

1. Mount coverslip with a drop of mounting medium.
2. Seal coverslip with nail polish to prevent drying and movement under microscope.
3. Store in dark at -20°C or 4°C.