• Jon Epstein,
  MCRC Director
• Michael Parmacek,
  Penn CVI Director
• Ed Morrisey
• Mark L. Kahn
• Peter Gruber
• Vic Patel
• Ken Margulies
• Cardiac Myocyte Core
• Histology and
  Gene Expression Core
• Microscopy Core
• Physiology and
  Microsurgery Core
• Plasmid Core
• Transgenic and
  Knockout Core
• Information for Staff
• Useful Links
• Search MCRC
• Penn CVI
• Penn CVI Seminars

Mouse Anti-_BrdU in paraffin embedded sections

ABC-HRP

1. Prewarm 2N HCl at 37 C. and prewarm 0.1M Tris PH 7.5.
2. Deparaffinize 20minX2. Rehydrate slides to ddH₂O.
3. Incubate slides in 2N HCl at 37 C for 15 min.
4. Rinse in ddH₂O 5min X 4.
5. 0.05% Trypsin in 0.1M Tris PH 7.5 at 37 C for 15 min.
   2.5% trypsin 1ml + 29ml in 0.1M Tris PH 7.5
6. .Wash in PBS 5min X 2.
7. Block endogenous peroxidase with 3% hydrogen peroxide in PBS) for 15 min.
8. Wash in PBS 5min X 3.
9. Block non-specific proteins with 10% normal goat serum in 0.5% PBST (PBS+0.5% Tween 20) for 30-60 min at RT.
10. Incubate with mouse anti BrdU 1:200---1:400 in Blocking buffer (5% goat serum in 0.1% PBT ) O/N at 4 C.
11. Wash slides with PBST 5min X3.
12. Incubate with 1:200 Biotinylated goat anti-mouse IgG (Vector) 1 hour at RT.
13. Make up ABC ( 30min before use) ABC 2.5ml (1:50)PBS+ 1drop of A + 1drop of B( 1drop=50ul )
14. Wash slides in PBST for 5minX3.
15. Incubate with ABC for 45 min.
16. Wash in PBSTfor 5min X3.
17. Wash in 0.1M Tris pH 7.5.( 0.1M Tris 7.5 & 0.3 M NaCl )
18. Color developing:
    DAB: One set tablets (Sigma) to 5ml ddH₂O.
19. Add DAB to slides for 2-10min.
20. Wash with ddH₂O several times.
21. Counter-stain with Hematoxylin for 1 min.
22. Wash and dehydrate in air. Mount the slides with mounting medium.

» Top