General Procedure for Paraffin Sections
(PCNA, mouse tissue as an example)
PROCEDURE:
1. Deparaffinize and hydrate samples to water.
2. Antigen Retrieval: Place slides in water, Microwave them for 5 min at full power. Allow to cool for 15 min.
3. Individually remove slides from water, dry around the tissue, circle tissue with a pap pen, add PBS to tissue and place in the humidity chamber. Rinse two more times with PBS.
4. Add 3% H202 in methanol for 12 min to block endogenous peroxidase activity.
5. Wash with PBS 3 time, 3 min for each time.
6. Add protein blocking solution (5% normal horse serum, + 1% normal goat serum in PBS) incubate 20 min.
7. Remove blocking solution and add primary antibody (PCNA-PC10, Dako Inc, cat. # M0879) diluted in protein block solution, incubate 2-3 hours at room temperature or overnight 4ºC.
8. Wash with PBS 3 time, 3 min for each time.
9. Incubate with protein block solution for 10 min.
10. Remove protein blocking solution and add secondary antibody (rat anti-mouse IgG2a HRP,Serotec) diluted in protein blocking solution, incubate 1 hour room temperature.
11. Rinse with PBS 3 time, 3 min for each time. followed by a brief rinse with PBS with Brij ( 1 drop of brij in 50 ml of PBS, pH 7.6)
12. Incubate with chromogen DAB solution for 5-10 min, check color development microscopically.
13. Wash with distilled water (d-water) 3 time, 3 min for each time, followed by a brief rinse in d-water with Brij (1 drop of brij in 50 ml of d-water)
14. Counterstain with Gill’s hematoxylin a few seconds (7-10 sec). Wash with d-water.
15. Add PBS 1 min to blue the nuclei.
16. Wash with d-water 3 time, 3 min for each time..
17. Dry slides or allow to air dry and mount with universal mount.