[Histonet] Re: Need assistance with in situ protocol!!! (LONG)

From:"Johnson, Teri"

Sorry it's taken me so long to reply, I've been unsubscribed for a
couple weeks!

Two things I think would help. Proteinase K digestion is optimal when
you can limit the time in the enzyme by altering concentrations.  We use
5 ug/ml for 7 minutes at 37 degrees C on our paraffin sections (7
microns thick), and that works fine for us.  This might help keep your
tissue on the slides a bit better.  Additionally, we use the secondary
antibody (sheep anti-dig) at 1:2000 dilution overnight at room
temperature.  I think your secondary antibody concentration is too
strong and that might be causing some of your problems.

Also do a blast search on your sequence and see if perhaps both the
sense and anti-sense are recognizing something in your sample (specific
binding).  As for commercial RNA probes, I haven't found any.  You might
try finding a sequence to GapDH or actin for positive control.

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110

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