Contents
Overview
R&D Systems provides monoclonal, polyclonal and biotinylated antibodies for immunohistochemical use. 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 tested by R&D Systems' Immunohistochemistry 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.The 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. Due to accumulation of autofluorescent pigment lipofuscin in primate and human neuronal tissues, the use of fluorescent probes, for example, FITC or Cy3 is not recommended. Protocols using chromogens (e.g., DAB, AEC or immunogold-silver staining) may be used instead. For Research Use Only.
Materials
Primary antibodies
- Unlabeled or biotinylated antigen-affinity purified polyclonal antibodies R&D Systems' AF or BAF series or selected monoclonal antibodies
Secondary antibodies
- Donkey anti-goat Cy3-conjugated antibodies (Cat# 705-165-147; Jackson ImmunoResearch Laboratories) or equivalent
- Donkey anti-chicken Cy3-conjugated antibodies (Cat# 703-165-155; Jackson ImmunoResearch Laboratories) or equivalent
- Donkey anti-mouse FITC conjugated antibodies (Cat# 715-095-151; Jackson ImmunoResearch Laboratories) or equivalent
- Donkey anti-goat FITC conjugated antibodies (Cat# 705-095-147; Jackson ImmunoResearch Laboratories) or equivalent
- Streptavidin conjugated to Cy3 (Cat# 016-160-084; Jackson ImmunoResearch Laboratories) or equivalent
- Streptavidin conjugated to FITC (Cat# 016-090-084; Jackson ImmunoResearch Laboratories) or equivalent
Buffers and additional supplies
- Fixation Buffer: 4% formaldehyde (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
- Avidin-Biotin Blocking Kit: (SP2001, Vector Labs)
- Wash Buffer: 0.1 M PBS, pH 7.4
- Mounting Media: anti-fade mounting medium i-BRITE Plus
(SF40000, Neuromics, Inc.)
Sample Preparation
- 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 - -70° C.
Tissue Staining
- 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 nonspecifically avidin should be treated with Avidin-Biotin Blocking Kit, according to manufacturer's instructions. Do not employ avidin blocking step when using biotinylated primary antibodies.
- 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.
- Incubate with the secondary antibody. Apply Streptavidin conjugated to Cy3 if a biotinylated antibody was used in step 5.
- Wash slides 3 times for fifteen minutes each in Wash Buffer.
- Mount with an anti-fade mounting media.
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