|  Turk
Lab Standard Operating Procedure (SOP) for Immunohistochemical Staining
for 11β hydroxysteroid dehydrogenase type 1
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 withTween 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 = rabbit polyclonal anti-rat 11β
hydroxysteroid dehydrogenase type 1 (source Dr. Zymunt Krozowski,
Baker Heart Research Inst
http://www1.baker.edu.au/flash.html, Melbourne, Australia). The antibody can be used on formalin-fixed
paraffin-embedded tissue using positive control adrenal gland, ovary,
testis (Figure 1) or adipose tissue (Figure 2).
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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