Day 3: Post-antibody washes
- Wash 3 times for 5 minutes each with TBST at room temperature.
- Wash 5 times for 1 to 1.5 hours in TBST at room temperature.
- Wash overnight in TBST at 4°C with gentle rocking.
Day 4: Color development
- Wash 3 times in NTMT for at least 10 minutes each at room temperature.
- Remove NTMT, add reaction mix (125 mg/ml BCIP and 250 m g/ml NBT in NTMT) and allow the reactions to develop at room temperature with gentle rocking. Throughout the development reaction, keep samples covered with aluminum foil to protect them from light and check them periodically to monitor the progress of the reaction.
- When the reaction is judged complete, wash with PBS or PBT and post-fix in 4% paraformaledehye/ 0.1% glutaraldehyde.
- Wash two to three times in PBS.
Solutions
All solutions should be made with high-quality reagent grade water that is RNase-free. (Note: We routinely use water directly from our purification system without DEPC treatment.) In addition, all solutions containing Tween-20 should be filtered and kept sterile.
10X PBS (1 liter):
PBT:
Hybridization solution:
Solution I:
Solution III:
Sheep serum:
10X TBS (for 1 liter):
TBST:
NTMT:
200X NBT:
200X BCIP:
