05/25/04
DAB Detection of HRP
DAB is a nasty known carcinogen. Treat it as if it were radioactive material ? glove changes, in the hood, bench paper ? to minimize your exposure.
1) Pre soak sample in DAB 5 minutes.
DAB is stored at ?20?C at 40mg/ml. Thaw at 37?C to make sure it is completely thawed and in solution before using.
Put 2ml 0.1M PO4 pH7.3 plus 25ml 40mg/ml DAB into two 5ml snap cap tubes, and mix by pipetting up and down.
Add one tube?s worth of PO4-DAB mixture to embryos, and let sit 5 minutes.
Use these 5 minutes to get everything set up at the microscope ready for immediate detection: dish, bench paper, transfer pipette
2) Add 2.5ml 3% peroxide to the other 2ml PO4 buffer plus DAB tube. Mix by pipetting up and down, and add to embryos.
Peroxide is stored at 4?C on Cecilia?s shelf.
3) Watch staining develop. The reaction begins immediately and goes very quickly!
4) To stop the reaction:
Take off the DAB-peroxide solution (put back into snap cap tube it came from)
Quickly rinse 3X with 0.1M PO4 Buffer
Transfer embryos to new eppi-tubes and store in PBST
To Clean up:
Use bleach to decontaminate everything you used, and all waste belongs in the hood .
