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)
wrote:

> 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,
Nottingham.
NG7 2UH



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