Materials & Methods > Core B > Immunohistochemical Staining for monocyte chemoattractant protein-1 (MCP-1)
 

Turk Lab Standard Operating Procedure (SOP) for Immunohistochemical Staining for monocyte chemoattractant protein-1 (MCP-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 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-rat MCP-1 (source PeproTech Inc, catalog # 500-P76) 1:100 dilution. The antibody can be used on formalin-fixed paraffin-embedded tissue using positive control coronary artery.

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

 

Material List (top)

1. Surgipath X-tra Slides® 00210

3. Black and Decker HS2000 vegetable steamer

3. Dako Target Retrieval solution S1699

5. Dako S3306

6. A/B block Vector

8. Rabbit polyclonal anti-rat MCP-1

10. Dako, LSAB+ Kit, K0690

12. Streptavadin, Dako , LSAB+ Kit K0690

14. DAB Dako, K3466



Literature (top)

Monocyte chemotactic protein-1, -2, and -3 are distinctively expressed in portal tracts and granulomata in primary biliary cirrhosis: implications for pathogenesis.

Monocyte chemoattractant protein-1 deficiency is protective in a murine stroke model.

17 beta-estradiol attenuates intimal hyperplasia and macrophage accumulation with a reduction in monocyte chemoattractant protein 1 expression in a vein graft model.

Increased monocyte adhesion to aortic endothelium in rats with hyperhomocysteinemia: role of chemokine and adhesion molecules.

Association between plasma levels of monocyte chemoattractant protein-1 and long-term clinical outcomes in patients with acute coronary syndromes.

C-reactive protein correlates with macrophage accumulation in coronary arteries of hypercholesterolemic pigs.