dig labelled Riboprobe whole mount insitu?

Dan Oerther doerther at nwu.edu
Fri Oct 7 12:46:53 EST 1994


Netters-
   We are attempting to use dig labelled riboprobe to detect the presence
of a transcription factor in whole mount insitus of MOUSE embryos
e9-e10.5.  So far, we have been unable to reproduce the pattern we would
expect from our control probes.  I believe that we are having a problem
with trapping of the riboprobe and possibly with insufficient specific
hybridization.  The control probes (sense and alpha-sense pFlk-1) are
approx 1 Kb, which we hydrolyze to a 200bp smear.  Hybridization is
carried out at 70dC O/N in 50%formamide with 1uG of probe in 1uL soln(very
stringent - perhaps too stringent?)  The riboprobe washes are done in 2uL
50%formamide, 2xSSC, 1%SDS for 3x30min at 70dC in 2uL soln.  Additionally,
after this initial wash, we treat the embryos with 10uG/uL RNase A in an
appropriate buffer.  Then wash again in soln above.  Our standard
preparation of the Mouse embryos is to dissect into RNases free PBS- then
go through 4%paraformadhyde fix, 3%H2O2 treatment, 15min 10uG/mL
Proteinase K treatment at 55dC, then refix in
4%paraformaldhyde/glutaraldhyde.
   My specific questions are for those who have had experience with Mouse
embryos (Not to ignore the advice of Drosophilia experts, but in this case
the tissues may react rather differently - but please offer any advice!!).

   1) are my hybridization conditions too stringent?
   2) what do you use to remove excess/nonspecific riboprobe i.e. washes?
   3) what levels of Proteinase K treatment do you recommend?
   4) any other advice - besides Give Up, this method will never work.

Thanks in advance,
Dan

P.S. feel free to e-mail response or post to bionet.methds-reagnts.  If I
get a number of e-mails I will post a summary to the group.


Dan Oerther
Northwestern University Evanston, IL
Dept of Biochemistry
doerther at nwu.edu

-- 
Dan Oerther
Departments of Civil Engineering and Biochemsitry
Northwestern University
Evanston, Illinois

doerther at nwu.edu



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