In situ hybridization question
phyllis.mann at tufts.edu
Thu May 31 11:41:10 EST 2001
I am doing ISHH using 33P labeled-riboprobes on rat brain tissue and I'm
having a problem during the development of the slides. I dip each slide
in NBT2 emulsion (diluted 1:1 in water), let dry in the dark standing up
for 3 hours and then store in slide boxes at 4 degrees C. Approx. 2
weeks later I develop. 4 minutes D19 (15C), 10 dips in water (stop,
18-20C), 10 minutes in fixer (18-20C), then 30 seconds of running water,
then drying in alcohols. After I counterstain, I usually have a thin
film of emulsion on the slides. Or what I think is emulsion, it looks
milky-white. I've tried variations on the development and it still the
same. In other labs that I have visited they do it quite similarly
but do not have the white film on their slides. Any suggestions?
Thank you very much in advance.
Phyllis E. Mann, Ph.D.
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