In situ hybridization question

Phyllis Mann phyllis.mann at
Thu May 31 11:41:10 EST 2001

Hi All,

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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