Enzymatic Protocol 1.1


R&D Systems provides monoclonal, polyclonal and biotinylated antibodies for immunohistochemical applications. Antigen affinity-purified polyclonal antibodies are recommended for immunohistochemistry because they are 10-fold more potent than the traditional protein G affinity-purified polyclonal antibodies. The use of analyte-specific affinity chromotography greatly reduces or totally eliminates non-specific immunoglobulins. The benefits of using antigen-affinity purified polyclonal antibodies are increased staining intensity and reduced non-specific staining.

The following protocol has been developed and optimized by R&D Systems’ Immunohistochemical Laboratory. R&D Systems’ antigen affinity-purified polyclonal and monoclonal antibodies have been used on frozen rat brain tissues as well as on paraffin-embedded human tissues. This protocol may need to be modified depending on the type of tissue used. Individual investigator should determine optimal working dilution of antibodies. If using R&D Systems’ primary antibodies, refer to product specification sheets to obtain approximate working dilutions. For all other reagents, follow manufacturer’s instructions. For Research Use Only.


Primary Antibodies

  • Unlabeled or biotinylated antigen< affinity-purified polyclonal antibodies (R&D Systems’ AF or BAF series) or selected monoclonal antibodies

Secondary Antibodies

  • Biotinylated donkey anti-goat IgG (Cat# 705-065-147; Jackson ImmunoResearch Laboratories)

Buffers and Additional Supplies

  • Fixative: 4% formaldehyde (Catalog # P6148; Sigma) and of 14% (v/v) saturated picric acid in 0.16 M phosphate buffered saline (PBS), pH 6.9
  • Sucrose Solution: 10% sucrose solution in 0.1 M PBS (pH 7.2)
  • Incubation Buffer: 0.1 M PBS, pH 7.4 containing 1% bovine serum albumin, 1% normal donkey serum, 0.3% Triton X-100 and 0.01% sodium azide
  • Wash Buffer: 0.1 M PBS, pH 7.4
  • Avidin-Biotin Blocking Kit: (SP-2001, Vector Labs)
  • Vectastain Elite ABC-Peroxidase Kit: (Cat# PK-6105; Vector Labs)
  • Substrates: AEC (Cat# SK-4200; Vector Labs), DAB (SK-4100), VIP (SK-4600), NovaRED (SK-4800, Vector Labs)
  • Mounting Media: Use an aqueous or a non-aqueous mounting media of choice

Sample Preparation (Frozen Tissues)

  1. Fix tissue by vascular perfusion with 500 - 700 mL Fixation Buffer.
  2. Perfuse tissue with 400 mL Sucrose Solution.
  3. Cut tissue using a cryostat at a nominal thickness of 5 - 15 µm and thaw-mount onto histological slides.
  4. Dry slides with tissue sections for 30 minutes on a slide warmer at 37° C.
  5. Store slides in a freezer at -20 to -70° C.

Tissue Staining: Chromogenic

  1. Thaw slides at room temperature for 10 minutes.
  2. Rehydrate the slides with Wash Buffer for 10 minutes.
  3. Drain excess Wash Buffer.
  4. Tissue suspected of binding avidin and biotin should be treated with Avidin-Biotin Blocking Kit, according to manufacturer's instructions.
  5. Apply primary antibodies diluted in Incubation Buffer and incubate at 4°C for 24 - 72 hours.
    Note: A negative control and/or an isotype matched control should be performed, which will identify non-specific binding from the secondary antibody. A negative control is the incubation buffer with no primary antibody. An isotype control may be employed when using monoclonal antibodies.
  6. Wash slides 3 times for fifteen minutes each in Wash Buffer. Proceed to step 9 if a biotinylated antibody was used in step 5.
  7. Incubate with the secondary antibody for 30 - 60 minutes at room temperature. Apply biotinylated secondary antibody if0 an unlabeled primary antibody was used.
  8. Wash slides 3 times for fifteen minutes each in Wash Buffer.
  9. Apply Vectastain ABC Elite Kit according to manufacturer's instructions.
  10. Rinse slides once in Wash Buffer.
  11. Prepare and apply Substrate of choice followed by a counterstaining of nuclei.
  12. Wash tissue according to manufacturer's instructions specified for the Substrate.
  13. Use an aqueous or a non-aqueous mounting media suitable for the Substrate.

Note: Alcohol washes out certain substrates, therefore, follow manufacturer's specifications for Substrate to prevent wash out.