VEGF

1.    Cut paraffin sections at 5m.

2.    Heat immobilize sections in a 60C for 30 min.

3.    Deparaffinize in 3 changes of xylene for 5 min each.

4.   Hydrate in series of graded ethanols starting with 2 changes of absolute, one 95%, and ending with one 70%, all for 5 min each.

5.    Rinse slides in distilled water for 2 min.

6.   Microwave antigen retrieval using Citra-plus {Biogenex} for 2 min on high power, check fluid level and add DI water if necessary. Microwave an additional 8 min. on 80% power. Let slides cool for 30 min.

7.   Rinse slides in 3 changes of distilled water.

8.   Rinse in 3 changes of PBS for 5 min each.

9.   Apply Normal Rabbit Serum 1:5 for 20 min at room temp (RT).

10. Drain excess serum and apply primary antibody mouse anti-VEGF {Santa Cruz} 1:20 for 1 hr. at RT.

11.  Rinse in 3 changes of PBS for 5 min each.

12. Apply biotinylated Rabbit-Mouse secondary antibody 1:200 {Dako}. Incubate for 30 min at RT.

13.  Rinse in 3 changes of PBS for 5 min each.

14.  Apply conjugated HRP - Streptavidin 1:1000 {Dako} in PBS. Incubate for 30 min at RT.

15.  Rinse slides in 3 changes of PBS for 5 min each.

16.  Apply stable DAB chromogen {Research Genetics} and incubate for 6 min at RT.

17.  Rinse slides in 2 changes of distilled water.

18.  Counterstain with dilute Harris hematoxylin 1:10 for 2 min.

19.  Rinse slides until clear in distilled water.

20.  Immerse slides in Scotts water for 1 min.

21.  Rinse slides in 2 changes of distilled water.

22.  Dehydrate, clear and mount with permount.