dig-labeled in situ

P. Jones/ P. Scotting mbzpj at unicorn.nott.ac.uk
Mon Jan 23 08:19:24 EST 1995

In article <pootanak-190195122852 at bi16.bc.edu>, pootanak at hermes.bc.edu (kp)

> Could anyone tell me the protocol for doing in situ hybridization on
> paraffin embeded tissue using dig-labeled cRNA probe.  I tried several
> times but couldn't detect any positive signals.  What I did was a standard
> protocol:
> a)deparafinization using either xylene or hemo-de
> b)hydration in graded ETOH.
> c)proteinase K treatment
> d)acetylation
> e)dehydration
> Thanks

We have published a brief protocol for this in the latest Boehringer
newsletter. Also for a similar description see Uwanogho et al, Mechanisms
of Development January 1995 edition (49, 23-36). Finally for a variety of
protocols there seem to be a lot of people beginning to do this stuff in
brains see Molecular Brain Research esp. vol 28, 122-128. Our personal bias
is to use a relatively standard protocol but the hyb solution is the same
as that used for whole mount in situs ie a bit thin without the usual
thickening components like dextran sulphate (we get more consistent results
this way).  Can supply detailed protocols on request.
Dr. Paul J. Scotting
Dept. of Biochemistry,
University of Nottingham,
Queen's Medical Centre,

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