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Lab Standard Operating Procedure (SOP) for Immunohistochemical Staining
for Smoothelin
Materials
Literature
1. Cut paraffin sections @ 4 microns and place on positively-charged
slides (Surgipath X-tra Slides® 00210)
2. Microwave slides briefly 1-2 min. on high power until paraffin
is just melted or bake in 60 degree C oven for 1 hour.
3. Deparaffinize , run slides to water and perform steamer (Black
and Decker HS2000 vegetable steamer) retrieval @ 90-100 C in Dako
Target Retrieval solution (S1699) for 30 min. with 20 min. room
temperature (RT) cooling time. Distilled water rinse several times.
4. Quench endogenous peroxidase with fresh 3% H202 for 5 min.
5. Rinse with distilled water several times and place in Tris with
Tween buffer (Dako S3306)
6. Block tissue with A/B block (Vector) for 15 min. each with a
Tris with Tween buffer rinse between and after.
7. Wipe around tissue and block with protein block (Dako X0909 )
for 10 min.
8. Drip protein block off slide and apply primary antibody for overnight
incubation. Primary antibody = monoclonal mouse anti-human Smoothelin
(Abcam, ab8969). 1:100 dilution, 30min at room temp. The antibody
can be used on formalin-fixed paraffin-embedded tissue. Positive
control aorta.
9. Next day place slides in Tris with Tween buffer and rinse once.
10. Wipe around tissue and apply secondary antibody for 30 min.
incubation at RT. (Dako, LSAB+ Kit, K0690)
11. Tris with Tween buffer rinse.
12. Apply label (Streptavadin, Dako , LSAB+ Kit K0690) for 30 min.
at RT
13. Rinse with distilled water several times.
14. Wipe around slides and apply diaminobenzidine (DAB Dako, K3466)
for 5-10 min., monitor slides and place in water when desired intensity
has been reached.
15. Rinse several times with distilled water.
16. Apply DAB enhancer (Dako, S1961) for 5 min. at RT.
17. Rinse several times with distilled water.
18. Give a 1 min. hematoxylin counterstain followed by 10 seconds
in 1% acid alcohol and 1 min. in ammonia water to blue.
19. Run through graded alcohols to xylene and coverslip with permanent
mounting media.
Last
GLP modification 7/1/04:
Jennifer
Casati
James R. Turk
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