| Turk Lab Standard Operating Procedure (SOP) for Immunohistochemical Staining for Fibronectin
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. Proteinase K can be used as pre-treatment
in place of steamer. 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 = rabbit polyclonal anti-human
fibronectin (source: DAKO , catalog # A0245 ), 1:800 dilution.
The antibody can be used on formalin-fixed paraffin-embedded tissue
using positive control block of canine subendocardial ischemia VMDL
05-02673-15
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. (Rabbit Envision+, Dako K4011 )
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.
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