In Situ Hybridizations of Seeds

mmelan at pattie.wellesley.edu mmelan at pattie.wellesley.edu
Tue Apr 20 18:54:30 EST 1993


Dear Networkers,
        Does anyone out there have experience with in situ hybridizations in
seed tissues?  (preferably Arabidopsis seed).  We are probing hydrated seed and
early germination stage seed.  We have modified the fixation and infiltration
steps of the paraffin sectioning to allow for slower movement of substances
into and out of the seed tissues.  For the most part, stained sections before
the hybridization steps look good (good cell morphology and good preservation
of nuclei and cytoplasm).  However, AFTER hybridizations are completed it
looks as if there are only cell walls left (i.e. very scant cytoplasm).  Has
anyone else out there experienced similar problems?  Are we not fixing the
tissue enough? (2% paraformaldehyde; 12-16 hrs)  Are we overdoing the ProteinaseK treatment? (1ug/ml; 37oC; 30 min)
        Any helpful suggestions will be appreciated.
===============================================================================
Melissa Melan                   (mmelan at lucy.wellesley.edu)
Department of Biological Sciences
Wellesley College
Wellesley, MA  02181
(617) 283-3137
===============================================================================



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