Immunohistochemistry (IHC) Protocol

Bake 4-5 um FFPE sections at 60 degrees C for 30-60 minutes. Deparaffinize in xylene, 2 changes for 5 minutes each. Rehydrate through 100 percent, 95 percent, and 70 percent ethanol, 2-3 minutes each, then rinse in water.

Place slides in citrate buffer pH 6.0 or Tris-EDTA buffer pH 9.0. Heat near boiling for 10-20 minutes using a pressure cooker, microwave, steamer, or water bath. Cool slides in buffer for 20 minutes, then rinse in TBS or PBS.

For HRP detection, incubate slides in 3 percent hydrogen peroxide for 10 minutes to quench endogenous peroxidase. Rinse 3 times in buffer.

Apply protein block, 5 percent normal serum, or 1-5 percent BSA for 20-60 minutes at room temperature. Drain but do not rinse if the detection system recommends leaving blocking protein on the section.

Dilute primary antibody in antibody diluent. Start with the datasheet-recommended IHC dilution or test 1:50, 1:100, 1:250, and 1:500. Apply 100-200 uL per section and incubate 1 hour at room temperature or overnight at 4 degrees C in a humidified chamber.

Wash 3 times for 5 minutes each in TBS-T or PBS-T. Keep slides covered with buffer and do not let tissue dry.

Apply HRP polymer, biotinylated secondary plus streptavidin-HRP, or another detection reagent according to the system instructions. Typical incubation is 20-30 minutes at room temperature. Wash 3 times.

Apply DAB or other chromogen for 1-10 minutes while monitoring signal under a microscope. Stop development by rinsing in water.

Counterstain with hematoxylin for 15 seconds to 2 minutes, blue in running tap water or bluing reagent, dehydrate through ethanol, clear in xylene, and mount with permanent mounting medium.

Cut 5-10 um cryosections and mount onto charged slides. Air dry 10-30 minutes at room temperature. Store slides cold if staining later.

Fix with cold acetone for 10 minutes at -20 degrees C, cold methanol for 5-10 minutes, or 4 percent paraformaldehyde for 10-15 minutes depending on the target. Rinse gently in PBS or TBS.

For HRP-based detection, quench endogenous peroxidase with 0.3-3 percent hydrogen peroxide for 10 minutes. For biotin-based detection, consider avidin/biotin blocking when tissue has high endogenous biotin.

Block 20-60 minutes with 5 percent normal serum or 1-5 percent BSA in PBS/TBS. Use serum from the secondary antibody host species when practical.

Dilute primary antibody in antibody diluent. Start with the datasheet-recommended frozen IHC dilution or test 1:50 to 1:500. Apply 100-200 uL per section and incubate 1 hour at room temperature or overnight at 4 degrees C.

Wash 3 times for 5 minutes. Apply secondary antibody, polymer detection, or fluorescent secondary antibody according to the detection system. Incubate 30-60 minutes.

For chromogenic detection, apply substrate until signal develops, rinse, counterstain, and mount. For fluorescent detection, protect from light, counterstain with DAPI if needed, and mount with antifade medium.

For FFPE tissue, deparaffinize, rehydrate, and perform antigen retrieval. For frozen tissue, fix according to target requirements. Rinse in PBS or TBS.

Treat autofluorescent tissues with a compatible quenching reagent when needed. Avoid quenchers that interfere with the planned fluorophores.

Use 0.1-0.3 percent Triton X-100 for intracellular access when compatible. Block with 5 percent normal serum or 1-5 percent BSA for 30-60 minutes.

Dilute primary antibody in blocking buffer. Start with the datasheet-recommended IHC/IF dilution or test 1:100, 1:250, and 1:500. Apply 100-200 uL per section and incubate 1-2 hours at room temperature or overnight at 4 degrees C.

Wash 3 times for 5 minutes. Add cross-adsorbed fluorescent secondary antibody at 1:500-1:1000 for 45-60 minutes protected from light.

Wash 3 times. Add DAPI if desired. Mount with antifade medium and coverslip without bubbles.

Acquire no-primary, single-color, and full-stain controls. Use identical image settings for samples that will be compared.

Use a known positive tissue or cell pellet and a known negative tissue when available. Include the experimental tissue only after positive control staining is working.

Test no retrieval, citrate pH 6.0, and Tris-EDTA pH 9.0 when tissue availability allows. Use the same section thickness and slide type for all conditions.

Dilute primary antibody across a small matrix such as 1:50, 1:100, 1:250, and 1:500, or follow datasheet starting ranges. Keep retrieval and detection constant while testing dilution.

If signal is weak, compare 1 hour at room temperature with overnight at 4 degrees C. Longer incubation can improve weak signal but may increase background.

If signal remains weak, increase polymer incubation within the detection system limits, use amplification, or extend chromogen development while monitoring background.

Increase wash time, reduce antibody concentration, change blocking buffer, shorten chromogen development, or add detergent to wash buffer if compatible with the tissue and detection system.

Once the best condition is chosen, stain all comparative samples together using the same retrieval, antibody dilution, incubation time, detection system, and development time.