1. Deparaffinize and rehydrate tissue section.
2. To reduce nonspecific background staining due to endogenous peroxidase, incubate slide in hydrogen peroxide for 10-15 minutes.
3. Wash 2 times in buffer.
4. If required, incubate tissue in digestive enzyme (or appropriate pretreatment).
5. Wash 4 times in buffer.
6. (Optional) Place slide in Ultra V Block or protein block and incubate for 5-10 minutes at room temperature to block nonspecific background staining. NOTE: With Ultra Block, do not exceed 10 minutes or there may be a reduction in desired stain.
7. Rinse (Optional).
8. Apply primary antibody and incubate according to manufacturer's protocol.
9. Wash 4 times in buffer.
10. Incubate slide with biotinylated secondary solution according to manufacturer's protocol.
11. Wash 4 times in buffer.
12. Incubate slide with Streptavidin Peroxidase solution for 10 minutes at room temperature.
13. Rinse 4 times in buffer.
14. Place slide in appropriate chromogenic substrate and incubate until desired reaction is achieved.
15. Counterstain and cover slip.
NOTE: This reagent is provided in bulk form for use with automated systems (including capillary gap methodologies) or in staining jars for repeated use. If the DIP method is employed, staining jar should be sealed and refrigerated between uses. Remove staining jars from the refrigerator 30 minutes prior to staining to allow reagents to come to room temperature.
The specificity and sensitivity of antigen detection is dependent on the specific primary antibody used.