Immunostaining for Factor VIII Related Antigen (endothelium):

1. Deparaffinize and rehydrate tissue sections. Rinse 3 times with PBS.

2. 0.1%Pepsin in 0.1N HCL in PBS incubated sections at 37°C for 40min.

3. Rinse with PSA 3x

4. 0.3% hydrogen peroxide in PBS, 30min. Then rinse in PBS 3x

5. 5% normal goad serum in PBS at RT for 1 hour.

6. Rabbit anti-Human Factor VIII Related Antigen diluted to 1:500 with 1% NGS in PBS, incubated at 4°C overnight. Rinse in PBS x3.

7. Goat Bionitylated anti-Rabbit IgG 1:200 in 1% NGS in PBS, incubated at RT for 1 hour. Rinse in PBS 3x.

8. ABC at RT for 1 hour. Rinse in PBS 3x. ( ABC solution: 5ml PBS with 1 drop A and 1 drop B, mix well)

9. DAB reaction: 2.5ml of distilled water + 1 drop Buffer + 2 drop DAB solution + 1 drop hydrogen peroxide. ~Needs 8 min to stop DAB reaction.

10. Rinse in distilled water 3x.

11. Counterstain and mount.