Question: in situ hybridization
Ward_Lab at tikal.biosci.arizona.edu
Thu Jul 25 16:04:23 EST 1996
For the past month, I've had a bit of difficulty trying to get in situ
hybridization to work on whole mount adult worms. I've been using an
adapted version of the embryo procedure from Seydoux and Fire (Ch 14 in
the "worm protocols" book). Now I'm interested in trying to do the in
hybridization on whole adult worms in suspension. SO- I'm about to try a
different in situ hybridization procedure and I was wondering if anyone
out there has any advice before I start. I'll be using the procedure
detailed by Albertson, et al. in Chapter 15 of the "worm protocols" book
(edited by Epstein and Shakes). One specific question I have about this
protocol is whether or not it's necessary to preform BOTH of the fixing
steps (first in formaldehyde, then in paraformaldehyde) if I won't be
freezing down the worms before I begin the Pretreatment steps.
As for the Seydoux and Fire protocol, I'd also be interested in any
suggestions on how I might improve on the penetration of my probe. I
don't think the freeze cracking is doing enough to get through the adult
cuticle, and the proteinase K digestion tends to digest the worms right
off the slides...
PLEASE SEND HELP!
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