| Turk Lab Standard Operating Procedure (SOP) for Immunohistochemical Staining for lipoprotein lipase (LPL)
Materials
Literature
 1.
Cut sections on cryostat @8-10 (or less) microns and place on silane
coated slides. (Surgipath X-tra Slides® 00210)
2. Let sections sit in incubator (37 C) overnight.
3. Proceed with staining or store airtight @-20C or lower.
4. Fix slides in acetone at RT for 10 min. and air dry.
5. Place slides in Tris buffer for 5 min. (Dako S3306)
6. Wipe around tissue and block with protein block (Dako X0909 )
for 10 min.
7. Block tissue with A/B block (Vector) for 15 min. each with a
Tris withTween buffer rinse between and after.
8. Drip protein block off slide and apply primary antibody for overnight
incubation. Primary antibody = rabbit polyclonal anti-human
lipoprotein lipase (LPL) (source: Dr. Brunzell ), 1:800 dilution.
The antibody can be used only frozen sections using porcine heart
as positive control.
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 twice.
12. Quench endogenous peroxidase with .3% H202 with sodium azide
(Dako) for 5 min.
13. Tris with Tween buffer twice.
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. Give a 1 min. hematoxylin counterstain followed by 10 seconds
in 1% acid alcohol and 1 min. in ammonia water to blue.
17. Run through graded alcohols to xylene and coverslip with permanent
mounting media.
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