Due to an overwhelming interest in getting the methods to fix larvae=20
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=
Thanks again and have a nice fly-day!.............Leif Sondergaard
Here follows the information I recieved.
Aloisia (Alice) Schmid
Howard Huges Medical Institute/Morrill Hall
University of Illinois
505 S. Goodwin Ave, 618 Morrill Hall
Urbana, IL 61801
Email: A=A9schmi at ux1.cso.uiuc.edu
Yes, I used to do this frequently. I fixed in Carnoy's fixative,=20
which is nasty but works well. I essentially poked a single hole in the=20
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=20
thatCarnoy's had picric-acid and I think ether and I don't remember what=20
else,but it is described in any classic entomology text. As soon as the=20
larvae are plopped into the Carnoy's they writhe rather
pathetically and then turn a milky white. After fixing, I dehydrated and=20
cleared as for any tissue,mounted in permaplast plus from Fisher and=20
sectioned to 8 um thicknesses. I don't know how you would proceed if you=20
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=20
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=
However, I used to use disposable knives for this
purpose and this worked well. I preferred using the paraffin method,
If you have any other question, I would be happy to try to help!
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 at 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=20
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=20
LindaRestifo (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=20
thesame. If you wish our protocol, just send me your address or if it is=20
very urgent your FAX number.
lraftery at cbrc.mgh.harvard.edu
Back in the olden days of S 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=20
lraftery at cbrc.mgh.harvard.edu and I will dig up my notes on temperature,=20
because I remember the sectioning temp for larvae was different than=
bachase at cwis.unomaha.edu
I make a slight tear using fine forceps, while holding the animal under=20
freshly made Carnoy's fixative. Fix for one hour at room temperature. =20
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=