We are doing In Situ PCR with dig/anti-dig system, with a final stain with DAB (Diaminobenzidine - Vector) but we are getting more background than expected at the end of the experiments. We are quenching endogenous peroxidase with 3%H2O2 in methanol, blocking of the tissue is done with BSA1%, 2xSSC per 15 mins at 37ºC. Primary antibody (anti-dig, 1/200) per one hour at RT, and finally detection with DAB (Vector) for 5-10 mins. Negative control of reaction (no taq, no primer) shows completely white; complete reaction (PCR mix with taq polymerase, primers, digoxigenine, 1%BSA, dNTPs) we think is showing up a brown color not necessary associated with a positive sample. Thanks
- How long do you quench for with Hydrogen peroxide ?
- You might find that incubating for upto 30 min will help
- That said your negative control is completely white so I'm not sure
- You could also try blocking with 5% BSA for 1 hour at room temp
- Finally try alternative sources of your Taq reagents (with higher grades of impurity) and decratinely include RFLP or better still Page purified primers rather than standard desalted if you are not already doing that
Dear Laurence, I’m quenching for 30 mins. Increasing the time and concentration of blocking with BSA worked better. Thank you for your suggestion
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