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fixing larvae

Leif Soendergaard lsunicph at BIOBASE.DK
Fri Apr 12 02:40:08 EST 1996

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.


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 at 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,
If you have any other question, I would be happy to try to help!



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 
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.



Laurel Raftery
lraftery at 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 at 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.



Bruce Chase
bachase at 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!


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