From owner-drosophila@net.bio.net Thu Apr 11 23:00:00 1996 Path: biosci!BIOBASE.DK!lsunicph From: lsunicph@BIOBASE.DK (Leif Soendergaard) Newsgroups: bionet.drosophila Subject: fixing larvae Date: 12 Apr 1996 00:40:08 -0700 Organization: BIOSCI International Newsgroups for Molecular Biology Lines: 112 Sender: daemon@net.bio.net Distribution: world Message-ID: <9604120738.AA16094@biobase.dk> NNTP-Posting-Host: net.bio.net Hi all, Due to an overwhelming interest in getting the methods to fix larvae forewarded I will post the information I recieved from different people to the news group. In case of use please quote the person who gave the information. Thanks again and have a nice fly-day!.............Leif Sondergaard Here follows the information I recieved. From: Aloisia (Alice) Schmid Howard Huges Medical Institute/Morrill Hall University of Illinois 505 S. Goodwin Ave, 618 Morrill Hall Urbana, IL 61801 Email: A-schmi@ux1.cso.uiuc.edu Yes, I used to do this frequently. I fixed in Carnoy's fixative, which is nasty but works well. I essentially poked a single hole in the larvae to help the permeabilization along, but with Carnoy's fixative even that isn't strictly necessary. And strangely, I never had trouble getting a beautiful in situ, despite the harsh chemicals in the fix. I remember thatCarnoy's had picric-acid and I think ether and I don't remember what else,but it is described in any classic entomology text. As soon as the larvae are plopped into the Carnoy's they writhe rather pathetically and then turn a milky white. After fixing, I dehydrated and cleared as for any tissue,mounted in permaplast plus from Fisher and sectioned to 8 um thicknesses. I don't know how you would proceed if you wanted to do whole mounts, but there is probably a protocol for that too. Alternatively, you can simply freeze and mount in OCT and section on the cryostat. That worked fine too, but is a little harder on your knife....the cuticle is in no way cleared and presents a more abrasive cutting surface. However, I used to use disposable knives for this purpose and this worked well. I preferred using the paraffin method, however. If you have any other question, I would be happy to try to help! =A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9 From: Helen Benes Department of Biochemistry/Molecular Biology University of Arkansas for Medical Sciences, Slot 516 4301 West Markham St. Little Rock, AR 72205 Tel.: (501) 686-5782 FAX: (501) 686-8169 E=A9mail: hbenes@biomed.uams.edu The most common method for paraffin embedding for in situ hybridization is to use 4% paraformaldehyde in 0.1 M Na phosphate, pH 7.2. The fixative should be made fresh and stored for up to a week maximum at room temperature. It is desirable to make small slits in the larvae, at the anterior and posterior ends to allow the fixative to penetrate well. Leave larvae in fixative (in a vial) over night at 4 C. Then one proceeds with EtOH dehydration and routine embedding. If you wish, I can give you a detailed protocol we have obtained from Linda Restifo (Univ. of Arizona), and have used ourselves successfully (with 35S-UTP-labeled riboprobes) and published in Devel. Biol. 142:138-146 (1990). We are currently modifying the protocol for non-radioactive digoxigenin-labeled probes but the fixation and tissue processing is all the same. If you wish our protocol, just send me your address or if it is very urgent your FAX number. =A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9 From: Laurel Raftery lraftery@cbrc.mgh.harvard.edu Back in the olden days of S[35] in situs, I did cryosections of larvae, and had xcellent results. The larvae are simply frozen in OCT mounting medium, and you need not fix. If you have access to a cryostat, and want to try this, contact me at lraftery@cbrc.mgh.harvard.edu and I will dig up my notes on temperature, because I remember the sectioning temp for larvae was different than embryos. =A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9 From: Bruce Chase bachase@cwis.unomaha.edu I make a slight tear using fine forceps, while holding the animal under freshly made Carnoy's fixative. Fix for one hour at room temperature. This seems to work quite well. I have also tried not making a slight tear, and have had good fixation, but (I think for reasons other than fixation) did not get good signal. I then wash in PBS and dehydrate, paraffin embed and section. I have not used cryostat sectioning. Good Luck! End =A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9=A9 .