I. Required material
Reagents
- Rabbit primary antibody
- ImmPRESS™ Reagent Anti-Rabbit Ig Peroxidase
- Peroxydase substrates
- Other related reagents
Buffers
- PBS : 10 mM sodium phosphate, pH 7.5, 0.9% saline
- others buffers
II. Experimentation length
- 20 or 50 mn of blocking
- 30 mn of sections incubation in ImmPRESS Reagent
- 20 or 45 mn of rinsing steps
- TOTAL : 1h10 to 1h40
III. Staining procedure for paraffin sections
1. Deparaffinize and hydrate tissue sections through xylenes or other clearing agents and graded alcohol series.
2. Rinse for 5 minutes in tap water.*
3. If quenching of endogenous peroxidase activity is required, incubate the sections for 30 minutes in 0.3% H2O2 in either methanol or water. Incubation times may be shortened by using higher concentrations of H2O2. If endogenous peroxidase activity does not present a problem, this step may be omitted.
4. Wash in buffer for 5 minutes.
5. When necessary, incubate sections for 20 minutes with ready-to-use (2.5%) normal horse blocking serum or blocking solution of choice.
6. Incubate sections with rabbit primary antibody diluted in appropriate antibody diluent solution.
7. Wash slides for 5 minutes in buffer.
8. Incubate sections for 30 minutes with ImmPRESS™ reagent.
9. Wash slides for 5 minutes in buffer.
10. Incubate sections in peroxidase substrate solution until desired stain intensity develops.
11. Rinse sections in tap water.
12. Counterstain, clear and mount.
* If antigen unmasking is required, perform this procedure after step 2, using citrate-based or high pH.
IV.Staining procedure for frozen sections
This procedure is generally appropriate for frozen sections, cell smears or cytocentrifuge preparations.
1. Sections are air dried.
2. Immediately before staining, fix sections with acetone or the appropriate fixative for the antigen under study.
3. Transfer slides into buffer.
4. 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 serum in PBS for 5 minutes; or 0.3% H2O2 in methanol for 30 minutes, or use other published methods (eg. Andrew, S. M., Jasani, B., Histochem J. 1987, 19, 426-30).
5. Follow steps 4-12 of the procedure recommended for paraffin sections.
IV.Search engine
- Primary antibodies
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