From owner-celegans@net.bio.net Wed Jul 24 23:00:00 1996 Path: biosci!bcm.tmc.edu!pendragon!news.msfc.nasa.gov!newsfeed.internetmci.com!in2.uu.net!news.Arizona.EDU!usenet From: Ward Lab Newsgroups: bionet.celegans Subject: Question: in situ hybridization Date: Thu, 25 Jul 1996 14:04:23 -0700 Organization: University of Arizona Lines: 22 Message-ID: <31F7E157.3700@tikal.biosci.arizona.edu> Reply-To: Ward_Lab@tikal.biosci.arizona.edu NNTP-Posting-Host: worm1.biosci.arizona.edu Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii Content-Transfer-Encoding: 7bit X-Mailer: Mozilla 2.0 (Macintosh; I; 68K) 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! Thanks, S. McKnight .