I. Required material
Reagents
- A primary antibody biotinylated
- Reagent A (Avidin DH) in orange-labeled small bottle
- Revelation kit VECTASTAIN® Elite ABC Reagent
Reagents not supplied
- Primary Antibody
- Buffer
- Hydrogen Peroxide
- Oxidizable Peroxidase Substrate
Buffers
- Xylene
- Alcooholic baths at 70%, 80% and 95%
- Hydrogen peroxide (H2O2)
-
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® Elite 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, use gentle H2O2 blocking to reduce the risk of antigen destruction or tissue loss:0.3% H2O2 in 0.3% Normal Sera in PBS for 5 minutes; or 0.3% H2O2 in methanol for 30 minutes, or use other published methods (e.g. Andrew, S. M., Jasani, B., Histochem J. 1987, 19, 426-430). If necessary, H2O2 treatment may also be performed after the biotinylated secondary antibody step.
- Follow steps 4-15 of the procedure recommended for paraffin sections.
IV. Search engines
- Primary antibodies
- Secondary antibodies
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