In situ hybridization

K J C 944 kjc944 at
Wed Jul 10 09:39:45 EST 1996

Dear Netters,
I'm getting ready to try whole mount in situ hybridization on mussel
larvae.  These larvae are, for the most part, less than 200 microns in
diameter.  While I understand the hybridization process, I am confused
about sample preparation.  I plan to fix the larvae in 4%
paraformaldehyde, 1X MOPS buffer.  My question is, how long do I fix my
larvae?  I know that fixation time is critical, but I have seen protocols
in which the fixation time is anywhere from 30 minutes to 48 hours.  I
don't have alot of larvae to mess around with and was wondering if there
is a rule of thumb for fixation time, depending on the size of the sample.
                                        My second question has to do with
dehydration.  Since I will not be imbedding my samples in parafin for
sectioning, is it still necessary to dehydrate?  Couldn't I just fix,
quench, and store in buffer at 4C until I'm ready to hybridize?

Thanks in advance!

 - Kathy

Kathryn Coyne
College of Marine Studies
University of DE
Lewes, DE  19958

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