I. Required material
Reagents
- A primary antibody
- A secondary antibody biotinylated
- Blocking serum (normal serum)
- Revelation kit (VECTASTAIN® ABC Reagent)
- Reagent A (Avidin DH)
- Reagent B (Biotinylated Horseradish Peroxidase H)
Buffers
- Xylene
- Alcoholic baths at 70%, 80% and 95%
- Hydrogen peroxide (H2O2)
- Buffer : 10mM sodium phosphate, pH 7,5, 0,9% saline (PBS)
Material
- Pipets and Pipet-aid
- Water bath
II. Experimentation length
- 30 mn of peroxydase blocking
- 30 mn of antigenic retrieval
- 1 h incubation of primary antibody
- 10 mn incubation of chromogen
- 2 h of rinsing steps and baths
- TOTAL : 4 hours
III. Protocol for paraffin sections
- Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series.
- Rinse for 5 minutes in tap water.
- If quenching of endogenous peroxidase activity is required, incubate the sections for 30 minutes in 0.3% H2O2 in methanol or water. Incubation times may be shortened by using higher concentrations of H2O2. If endogenous peroxidase activity does not present a problem, step 3 may be deleted.
- Wash in buffer for 5 minutes.
- Incubate sections for 20 minutes with diluted normal serum (large yellow-labeled bottle) from the species in which the secondary antibody is made. (In cases where non-specific staining is not a problem, Steps 5 and 6 may be deleted).
- Blot excess serum from sections.
- Incubate sections for 30 minutes with primary antiserum diluted in buffer. (If background staining occurs, dilutions of the primary and secondary antibodies may be made in buffer containing 1-2% of the appropriate blocking serum.)
- Wash slides for 5 minutes in buffer.
- Incubate sections for 30 minutes with diluted biotinylated secondary antibody solution (large blue-labeled bottle).
- Wash slides for 5 minutes in buffer.
- Incubate sections for 30 minutes with VECTASTAIN® ABC Reagent.
- Wash slides for 5 minutes in buffer.
- Incubate sections in peroxidase substrate solution until desired stain intensity develops.
- Rinse sections in tap water.
- Counterstain, clear and mount.
IV. Protocol for frozen sections
This procedure is generally appropriate for frozen sections, cell smears or cytocentrifuge preparations.
- Sections are air dried.
- Immediately before staining, fix sections with acetone or the appropriate fixative for the antigen under study.
- Transfer slides into buffer.
- If quenching of endogenous peroxidase is required, follow the procedure in step 3 of the paraffin section protocol. In some cases, especially when using monoclonal antibodies, the antigen may be destroyed by treatment with H2O2. In these cases, this treatment should be used only after incubation with biotinylated antibody.
- Follow steps 4-15 of the procedure recommended for paraffin sections.
After completion of the staining procedure, dilute working solutions should be discarded, and the containers washed with distilled water and stored together with the stock reagents in the kit box.
V.Search engines
- Primary antibodies
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