Vimentin

1. Cut paraffin sections at 5m and place on APES treated slides (Superfrost + or Surgipath X-tra)..

2. Heat immobilize in an 80C oven for 20 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. Rinse in 3 changes of PBS for 5 min. each.

7. Apply primary antibody Vimentin 1:100 {Biogenex) for 60 min. at room temp.

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

9. Apply Rabbit-Mouse biotinylated secondary antibody 1:200 {Dako} in PBS with 10% normal rabbit serum. Incubate for 30 min. at room temp.

10. Rinse in 3 changes of Tris Buffered Saline (TBS) for 5 min. each.

11. Apply conjugated alkaline phosphatase - Streptavidin 1:50 {Biogenex} in TBS. Incubate for 30 min. at room temp.

12. Rinse slides in 3 changes of TBS for 5 min. each.

13. Apply fast red chromogen {Biogenex} (1 tablet + 5ml naphthol buffer). Incubate for 10 min. at room temp.

14. Rinse slides in 2 changes of distilled water.

15. Counterstain with dilute Harris hematoxylin (1:10) for 2 min.

16. Rinse slides until clear in distilled water.

17. Immerse slides in Scotts water for 1 min.

18. Rinse slides in 2 changes of distilled water.

19. Mount with crystal mount or other aqueous mounting medium.

FAST RED IS AN ALCOHOL SOLUBLE CHROMOGEN!

DO NOT DEHYDRATE ALCOHOLS OR CLEAR IN XYLENE!