From owner-dros@hgmp.mrc.ac.uk Wed Jun 20 19:37:39 2001 Return-Path: Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id 1AA1017A5F for ; Wed, 20 Jun 2001 19:37:38 +0100 (BST) Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id 5A43B17A84 for ; Wed, 20 Jun 2001 19:37:35 +0100 (BST) Received: by mercury.hgmp.mrc.ac.uk (Postfix, from userid 6020) id 83ABF17A82; Wed, 20 Jun 2001 19:37:31 +0100 (BST) Received: from localhost (localhost [127.0.0.1]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id D1C9517BAE for ; Wed, 20 Jun 2001 17:16:47 +0100 (BST) Received: from mailbox2.ucsd.edu (mailbox2.ucsd.edu [132.239.1.54]) by mercury.hgmp.mrc.ac.uk (Postfix) with ESMTP id 4F9FD17A56 for ; Wed, 20 Jun 2001 17:16:29 +0100 (BST) Received: from oxmail.ox.ac.uk (oxmail2.ox.ac.uk [163.1.2.1]) by mailbox2.ucsd.edu (8.11.3/8.11.0) with ESMTP id f5KGGRA03587 for ; Wed, 20 Jun 2001 09:16:27 -0700 (PDT) Received: from news.ox.ac.uk ([163.1.2.7]) by oxmail.ox.ac.uk with esmtp (Exim 3.12 #1) id 15CkeY-0000Ln-00 for bionet-drosophila@moderators.isc.org; Wed, 20 Jun 2001 17:16:26 +0100 Received: (from news@localhost) by news.ox.ac.uk (1.2/8.6.12) id RAA15284 for bionet-drosophila@moderators.isc.org; Wed, 20 Jun 2001 17:15:54 +0100 To: bionet-drosophila@moderators.isc.org From: Helen White-Cooper Newsgroups: bionet.drosophila Subject: Re: imaginal disc in situs Organization: Oxford University, England Message-ID: <3B30DA92.A4B6AC7F@zoo.ox.ac.uk> References: <5.1.0.14.0.20010607180053.00aa0c60@imap.ndsu.nodak.edu> <9fqhre$cc2$1@uni00nw.unity.ncsu.edu> Mime-Version: 1.0 Content-Type: text/plain; charset=us-ascii; x-mac-type="54455854"; x-mac-creator="4D4F5353" Content-Transfer-Encoding: 7bit X-Trace: news.ox.ac.uk 993053754 15282 129.67.27.96 (20 Jun 2001 16:15:54 GMT) X-Complaints-To: newsmaster@ox.ac.uk NNTP-Posting-Date: Wed, 20 Jun 2001 16:15:54 +0000 (UTC) X-Mailer: Mozilla 4.77C-CCK-MCD {C-UDP; EBM-APPLE} (Macintosh; U; PPC) X-Accept-Language: en Date: Wed, 20 Jun 2001 19:37:31 +0100 (BST) Sender: owner-dros@hgmp.mrc.ac.uk Precedence: bulk We also get round the problem of tissue loss by doing all the steps after the second fixation in baskets. these are tissue culture inserts (falcon) with 8 micron mesh at the bottom that sit in 24 well tissue culture plates. You put solutions into the basket, and take them out from the well. To get round the background problem incubate in antibody overnight, 4 degree rather than room temp 2hrs. This means you can also increse the number and total time of the washes in hyb buffer, and is great for reducing background in testes. I do at least 6 washes, at least 3hrs total in hyb buffer (ie >6x30 min washes). good luck Helen Jim Mahaffey wrote: > Hi Mary, > > I can't help with the background problem, but can offer a solution to the > tissue loss. When doing antibody staining to imaginal discs, we leave the > discs attached to the inverted head. We just fix the whole inverted head > after removing much of the fat body etc. That way, the discs are not loose. > After staining, we remove the discs and place them on slides to view. > > Hope this helps, > > Jim Mahaffey > > "Mary Stewart" wrote in message > news:5.1.0.14.0.20010607180053.00aa0c60@imap.ndsu.nodak.edu... > > Hi, > > Can anyone help us with advice on in situs to imaginal discs? We made > > dig-labeled sense and antisense probes which worked great on embryos---no > > background with the sense probe. We also have anti-dig antibody that was > > preabsorbed to embryos and this worked great on embryos. When we tried > > these reagents on imaginal discs, we have a low, but unacceptable, level > of > > background with the sense probe. The antisense gives a stronger signal, > > but we're not comfortable with the results since we have some overall > > general staining with the sense probe as well as some staining that > > resembles what we see with the antisense (even though the antisense > > staining is more intense). Does anyone have some advice on how we might > > solve these problems? Also, any advice on how to get through all the > > handling steps and minimize the amount of tissue we lose? We lost a lot > of > > discs during all the handling and washing steps. Thanks for any help. > > Mary Stewart > > > > > **************************************************************************** > ** > > Mary Stewart > > Assistant Professor > > Department of Biological Sciences > > North Dakota State University > > Office: 701-231-8226 > > Lab: 701-231-6523 > > .