-
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 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.
4. Wash in buffer for 5 minutes.
5. Incubate sections for 1hour at 37℃with Blocking Serum
6. Blot excess Blocking Serum from sections.
7. Incubate sections for 1 hourat 37℃or Overnight at 4℃with primary antibody diluted in PBS buffer. (If background staining occurs, dilutions of the primary antibody may be made in buffer containing 0.1% of BSA, See Note 3, 5)
8. Wash slides for 3 minutes in PBST buffer.
9. Incubate sections for 1 hour at 37℃ with poly- HRP Goat Anti-Rabbit IgG.
10. Wash slides for 3 minutes in PBST buffer.
11. Incubate sections in DAB working solution until desired stain intensity develops. (See Note 2)
12. Rinse sections in tap water.
13. Counterstain, clear and mount.