larvae fixing

Leif Soendergaard lsunicph at BIOBASE.DK
Fri Apr 12 02:15:27 EST 1996


Hi all,
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=
=20
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=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=
=20
that isn't strictly necessary.  And strangely, I never had trouble getting a=
=20
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=
=20
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!

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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 at biomed.uams.edu


The most common method for paraffin embedding for in situ hybridization is=
=20
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=
=20
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.


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From:

Laurel Raftery
lraftery at cbrc.mgh.harvard.edu


Back in the olden days of S[35] in situs, I did cryosections of larvae, and=
=20
had xcellent results.
The larvae are simply frozen in OCT mounting medium, and you need not fix.=
=20
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=
 embryos.


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From:

Bruce Chase
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=
 Luck!

End
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