CD31 /Brdu Fluorescent Double Staining Protocol
Note: CD31 is widely used as an endothelical marker and Bromodeoxyuridine (Brdu) uptaking as a proliferating marker. When vascular endothelial cells go proliferation, they will take up Brdu to synthesize DNA if Brdu is available in their environment. So scientists put Brdu in cell culture medium or inject it into animal to let it incorporate into proliferating cells. In angiogenesis study, investigators can use CD31 antibody to label endothelial cells and use Brdu antibodies to label proliferating cells, by double staining, only proliferating endothelial cells can be indentified. This is an important technique in angiogenesis study.
CD31 Staining procedure
1. Fix slides in cold acetone for 5 min, followed by acetone:chloroform (1:1) for 5 min and acetone alone again for 5 min.
2. Wash in PBS solution 3 min for two times
3. Individually remove slides, dry around the tissue, care should be taken not to let the tissue dry, draw a circle around the tissue with a Pap pen, add drops of PBS to tissue and place in humidity chamber.
4. Block the samples with protein block solution (5% normal horse serum + 1% normal goat serum in PBS) for 20 min at room temperature.
5. Remove protein block and add primary antibody (rat anti-mouse CD31, PharMingen Inc. Cat. #01951A) diluted in protein block solution. Incubate overnight at 4ºC.
6. Wash with PBS 3 times, 3 min each time.
7. Block with protein block solution for 10 min
8. Remove protein block solution and add secondary antibody conjugated with Texas Red (goat anti-rat Texas-red, Jackson ImmunoResearch Labs, Inc.) diluted in protein block solution. Incubate 1 hour at room temperature. Start from this step, always cover humidity chamber with aluminum foil to protect slides from light.
9. Wash at least 3 times, 5 min each time with PBS and then wash with PBS with Brij (1 drop of brij in 50 ml of PBS pH7.6) for 3 min.
BRDU Staining procedure
10. Add 4% paraformaldehyde (diluted in PBS) for 10 min room temperature
11. Wash slides with PBS 3 times, 3 min each time.
12. Remove PBS and add 1% Triton-X-100 for 8 min at room temperature.
13. Wash slides with PBS 3 times, 3 min each time.
14. Incubate with 2N HCL in PBS 30 min at 37°C
15. Wash slides with PBS 3 times, 3 min each time.
16. Wash 0.1M Tris 2 times, 5 min each time.
17. Block with protein block solution for 10 min.
18. Incubate with Anti-Brdu (Anti-Bromodeoxyuridine monoclonal antibody, Becton-Dickinson, Cat# 7580)1-2 hr at room temperature.
19. Wash slides with PBS 3 times, 3 min each time.
20. Block with protein block solution for 10 min.
21. Remove protein block solution and add secondary antibody(Alexa Fluor 488 goat anti-mouse IgG, Molecular Probes) and incubate I hr at room temperature.
22. Wash slides with PBS 3 times, 3 min each time.
23. Counterstain with Hoechst 33342 (1 ug/ml in PBS) for 2 min to locate nuclei.
24. Wash slides with PBS 3 times, 3 min each time.
25. Mount with propyl gallate and a glass cover slip.