Immunohistochemistry (IHC) Protocol

Remove paraffin with xylene or substitute, then rehydrate through graded alcohols to water.

• Incomplete deparaffinization causes patchy staining.

Use heat-induced epitope retrieval in citrate, EDTA, or another validated retrieval buffer.

• Choose retrieval pH based on antibody datasheet or optimization.

Block endogenous peroxidase for HRP detection and block non-specific tissue binding.

• Use serum or protein blocker matched to the detection system.

Apply IHC-validated primary antibody at the recommended dilution and incubation time.

• Run positive and negative tissue controls.

Add detection reagent, develop chromogen, counterstain, dehydrate if required, clear, and mount.

• Stop chromogen development before background becomes excessive.

Bring frozen sections to the recommended temperature and fix if not already fixed.

• Use acetone, methanol, or paraformaldehyde only if compatible with the target.

Wash gently and block non-specific binding.

• Handle sections carefully because frozen tissue can detach or tear.

Incubate with antibody validated for frozen tissue or IHC where possible.

Use chromogenic or fluorescent detection matched to the experimental goal.

Counterstain appropriately and mount using medium compatible with detection method.

Use FFPE or frozen tissue preparation appropriate to the target and antibody.

Perform antigen retrieval if needed and block tissue background.

• Reduce autofluorescence if the tissue type requires it.

Use validated primary antibodies and confirm species compatibility for multiplex staining.

Use cross-adsorbed fluorophore-conjugated secondary antibodies and protect from light.

Mount with anti-fade medium and acquire images with channel-specific exposure settings.

Use a known positive tissue and a known negative tissue before interpreting experimental samples.

Compare retrieval buffer pH and time without changing every variable at once.

Test a small dilution series around the datasheet recommendation.

Run no-primary and isotype or negative reagent controls if appropriate.

Once signal and background are acceptable, keep retrieval, antibody dilution, incubation time, and detection time consistent.