BRDU PROTOCOL (ZYMED KIT + VECTOR DAB KIT, WITH MODIFICATIONS)
Zymed BrdU: Invitrogen #93-3943
Vector DAB: Vector #SK-4100
1. Dry sections on warmer at 60°C.
2. Deparaffinize/rehydrate sections:
 Xylene, 5 min x3
 100% EtOH, 1 min x3
 PBS, 5 min
3. Peroxidase quenching:
 25 ml 30% H2O2 to 225 ml absolute methanol
 RT, 10 min
4. Wash with 1X PBS 2 min x3 on rocking platform
5. Circle section with Pap-pen [Invitrogen #00-8877]
6. Trypsin digestion (1 part 1A + 2 parts 1B):
 Ex. 12 drops 1A to 24 drops 1B and mix well
 Incubate in moist chamber for 15 min at 37°C, in humidified chamber
7. Wash with dH20 2 min x3 on rocking platform
8. Denature w/ 2 drops reagent 2 for 30 min at RT, in humidified chamber
9. Wash with PBS 2 min x3 on rocking platform
10. Apply 2 drops Blocking solution (reagent 3). Incubate for 30 minutes at RT
11. Remove blocking solution, and apply 2 drops biotinylated mouse anti-BrdU (reagent 4).
Incubate 2 hours at RT, in humidified chamber
12. Wash with PBS 2 min x3 on rocking platform
13. Apply 2 drops of streaptavidin-peroxidase (reagent 5). Incubate 15 min at RT, in
humidified chamber
14. Wash with PBS 2 min x3 on rocking platform
Updated 4/21/15 by LM
15. Make DAB (use VECTOR DAB – much stronger!);
 To 5 ml DISTILLED H2O add 2 drops buffer sol. Mix well.
 Add 4 drops DAB stock sol., mix well
 Add 2 drops H2O2 substrate sol., mix well
 Protect from light and use within one hour.
 (optional) Add 2 drops Nickel sol, if NOT counterstaining with hematoxylin
*add 100 l and incubate for 10 minutes
16. Rinse well with dH2O.
17. Counterstain slides with 2 drops hematoxylin (reagent 7) for 5 minutes
18. Wash slides with tap water
19. Put slides into PBS until sections turn blue (~30 sec)
20. Rinse in dH2O
21. Dehydrate through ethanol and xylene,
22. Mount with Xylene-based mounting medium.
Updated 4/21/15 by LM