dig labelled Riboprobe whole mount insitu?

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

   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
   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,

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

More information about the Methods mailing list