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.
- Unlabeled or biotinylated antigen< affinity-purified polyclonal antibodies (R&D Systems’ AF or BAF series) or selected monoclonal 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)
- Fix tissue by vascular perfusion with 500 - 700 mL Fixation Buffer.
- Perfuse tissue with 400 mL Sucrose Solution.
- Cut tissue using a cryostat at a nominal thickness of 5 - 15 µm and thaw-mount onto histological slides.
- Dry slides with tissue sections for 30 minutes on a slide warmer at 37° C.
- Store slides in a freezer at -20 to -70° C.
Tissue Staining: Chromogenic
- Thaw slides at room temperature for 10 minutes.
- Rehydrate the slides with Wash Buffer for 10 minutes.
- Drain excess Wash Buffer.
- Tissue suspected of binding avidin and biotin should be treated with Avidin-Biotin Blocking Kit, according to manufacturer's instructions.
- 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.
- Wash slides 3 times for fifteen minutes each in Wash Buffer. Proceed to step 9 if a biotinylated antibody was used in step 5.
- Incubate with the secondary antibody for 30 - 60 minutes at room temperature. Apply biotinylated secondary antibody if0 an unlabeled primary antibody was used.
- Wash slides 3 times for fifteen minutes each in Wash Buffer.
- Apply Vectastain ABC Elite Kit according to manufacturer's instructions.
- Rinse slides once in Wash Buffer.
- Prepare and apply Substrate of choice followed by a counterstaining of nuclei.
- Wash tissue according to manufacturer's instructions specified for the Substrate.
- 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.