Immunohistochemistry Protocol

Date Published: July 18, 2023

Protocols for immunohistochemistry vary widely due to differences in antigens and their recognition by antibodies. Various fixation methods can uncover an epitope, destroy it, or make it less accessible. For successful results, it is crucial to try a number of different techniques and compare the signal obtained. The best negative control is pre-incubation of the antibody with an excess of the peptide or protein immunogen, although tissue known not to express the antigen is also commonly used. This protocol represents the basic avidin-biotin-peroxidase complex procedure recommended for proteins present in relatively high abundance.

Required Materials

Fixation

  1. Fix tissues in fixative (if needed) for 10 minutes. Larger tissue sections may require longer incubation periods. The fixation time will affect the characteristics of the section tissue and possibly subsequent antigen recognition.
  2. For paraffin-embedded tissue, incubate in two changes of fresh xylene for 5 minutes each. Rehydrate the tissue in a series of graded ethanol solutions as follows: 2 x 100%, 1 x 95%, 1 x 70% and 1 x 50%, for 3 minutes each. Rinse in distilled water.

Antigen retrieval (optional)

Antigen unmasking may be required if the analyte is cross-linked or modified in the tissue due to the fixation process. Typical antigen unmasking techniques utilize boiling, pressure cooking, or steaming tissue sections. Prepare 10 mM citrate buffer, pH 6.0. Boil slides in the citrate buffer in a microwave for 7 to 10 minutes. Wash slides in water for 3 minutes, then place in PBS.

Block non-specific binding sites

Cover tissue section with 5% Blocking Serum.

Primary antibody

  1. Dilute primary antibody in IHC diluent to the recommended dilution on the product specification sheet. The optional dilution must be determined by the investigator for each experiment.
  2. Use lint-free laboratory tissue to remove excess buffer from the slide surrounding the tissue. Circling the tissue section with a hydrophobic wax pen is optional, but helps confine antibodies to the tissue section.
  3. Cover the tissue section with the primary antibody solution. Incubate in a humid chamber for one hour at room temperature.
  4. Wash in three changes of PBS for 7 minutes each.

Secondary antibody

  1. Dilute the appropriate biotin-conjugated secondary antibody in IHC Diluent according to the recommendations on the specification sheet. The optimal dilution must be determined by the investigator for each experiment.
  2. Cover the tissue section with the secondary antibody solution. Incubate in a humidified chamber for 30 minutes at room temperature.
  3. Wash in three changes of PBS for 7 minutes each.

Blocking endogenous peroxidase (optional)

  1. Incubate the tissue sections for 5 to 10 minutes at room temperature in a 1:10 solution of 3% hydrogen peroxide in 100% methanol.
  2. Rinse in distilled water.
  3. Wash in PBS for 7 minutes.

Binding avidin-peroxidase conjugate

  1. Cover the tissues sections with the streptavidin-horseradish peroxidase conjugate diluted to the manufacturer’s recommendation for IHC.  The optimal dilution must be determined by the investigator for each experiment.
  2. Incubate for 30 minutes at room temperature in a humidified chamber.
  3. Wash in 3 changes of PBS for 7 minutes each.

Visualization with DAB

  1. Freshly prepare DAB solution by combining 1 mg DAB per mL of PBS.
  2. Pass DAB solution through filter paper into a clean test tube.
  3. Add 1 µL of 3% hydrogen peroxide per mL of DAB solution. Mix gently.
  4. Cover the tissue section with activated DAB solution.
  5. Incubate in a humidified chamber until color develops (~5-10 minutes).
  6. Stop the reaction by rinsing slides in distilled water.
  7. Wash in two changes of PBS for 5 minutes each.

Counterstain with hematoxylin

  1. Dip sections in hematoxylin solution (do not incubate).
  2. Rinse in several changes of cold tap water until the water runs clear.
  3. Leave slides in tap water for 5 minutes.
  4. Rinse in distilled water.

Dehydration and mounting

  1. Dehydrate the tissue sections in a series of graded ethanol solutions as follows: 50%, 70%, 95% and 2 x 100%, for 3 minutes each.
  2. Incubate in two changes of xylene for 5 minutes each.
  3. Mount with standard mounting glue of your choice and top with glass coverslips.

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