Hello,

  I am currently using Dig-labeled sense and as probes on fresh frozen
mouse brain cryosections.  I have been using anti-dig AP, but I find it
takes a long time to develop the signal with NBT/BCIP (this is a rare
mRNA transcript), and also find that the sections get much darker over
time (someone else brought this up on histonet recently).  I am
considering using Dako's anti-dig HRP, but I was wondering if (and where
in the protocol) I then needed to include a peroxidase blocking step.
Also, is it necessary to do a TSA amplification with anti-dig HRP, or
can I still get clean signal with just a DAB incubation? I would greatly
appreciate anyone's advice on this matter!

Thanks!
Anna Beaudin
Division of Nutritional Sciences
Cornell University
Ithaca, NY


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