1. Cut paraffin sections at 5µ and place on APES treated slides (Superfrost + or Surgipath X-tra).
2. Heat immobilize sections in an 80°C oven for 20 min.
3. Deparaffinize the sections in xylene 3 X 5 min. each.
4. Place the sections in absolute ETOH 2 X 5 min. each.
5. Hydrate in 95% ETOH for 5 min.
6. Hydrate in 70% ETOH for 5 min.
7. Rinse in dionized (DI) H2O for 2 min. Note: If sections were cut from carmine stained whole mount sections then destain in 0.4% lithium carbonate for 5 - 10 minutes followed by several DI H2O rinses. This procedure is designed for methacarn fixed tissue. However, If the tissue is formalin fixed perform microwave antigen retrieval prior to acid hydrolysis. All incubations are performed at room temperature.
8. Place slides in 2N HCL for 90 min.
9. Rinse in DI H2O for 2 min.
10. Place slides in 0.1M Na2B4O7 for 5 min (do not use on sections from Carmine stained whole mounts).
11. Rinse in DI H2O for 2 min.
12. Block endogenous peroxidase activity using 3% H2O2 for 5 min.
13. Rinse in DI H2O for 2 min.
14. Rinse slides in phosphate buffered saline (PBS) 3 X 5 min. each.
15. Apply anti-BRDU # 347580 (BD Immunocytometry Systems, San Jose, CA) 1:40, incubate for 1 hour.
16. Rinse slides in PBS 3 X 5 min. each.
17. Apply biotinylated Rabbit anti-Mouse # E464 (Dako, Carpinteria, CA) 1:200 in 10% Normal Rabbit Serum # X902 (Dako, Carpinteria, CA), incubate for 30 min.
18. Rinse slides in PBS 3 X 5 min. each.
19. Apply conjugated HRP streptavidin # P397 (Dako, Carpinteria, CA) 1:1000, incubate for 30 min. Note: remove an aliquot of Stable DAB from the -20°C freezer and place on bench top. The DAB must be equilibrated to room temperature prior to application in step 21.
20. Rinse slides in PBS 3 X 5 min. each.
21. Apply Stable DAB # 750118 (Research Genetics, Huntsville, AL ), incubate for 6 min.
22. Rinse slides in DI H2O 3 X 1 min. each.
23. Counterstain using hematoxylin. Use Mayers, Gill I or if performing image analysis on the CAS-200 image analysis system use dilute Harris hematoxylin (1:10) for 2 min.
24. Rinse until clear and blue in Scotts water for 1 min, rinse in DI H2O.
25. Dehydrate using a series of graded ethanols, starting with 95% ETOH, 2 X 2 min.
26. Dehydrate in absolute ETOH, 2 X 2 min.
27. Clear in xylene, 3 X 2 min.
28. Mount sections with Permount or other synthetic resin.