Re: [Histonet] sectioning bee's heads and mites
From time to time I have to section insects and it is difficult since
their little bodies are hard on the outside and processing makes them
harder. I recently found a procedure that might help to make life a little=20
easier when faced with sectioning these critters. It involves using Butanol=20
and ETOH. See if you can find a copy of "Manual of Basic Techniques in
Insect Histology" by Pedro Barbosa. In it is a procedure called Stiles'
N-Butyl Alcohol Technic. It is very time consuming - takes days but could
be worth it. I had to order in the book from another library since the
library here didn't have a copy and the book is out of print. Let me know
if you want the details of this procedure and I can scan the pages I copied=20
before returning the book and email them to you. Unfortunately I have not
used this procedure to tell you myself how it works because the project was=20
completed about the same time I found the book.
Also - another method that works OK: the investigator who brought the last=20
insect project to my lab fixed whiteflies in Penfix (Richard Allen
Scientific). He removed the wings and legs and continued fixing for 36 hrs.=20
Rinsed in 70% ETOH and removed any remaining wings and legs he missed the
first time. A tedious task for something as small as whiteflies! Glad he
was doing it!!! Can you just picture this guy sitting there looking through=20
the dissecting microscope pulling off their little legs and wings? Sounds
I processed on a MVP tissue processor, 15 min in each station starting at
70% ETOH then 80%, 2-95%, 3-100. All at room temp and vacuum used in the
last station of the 95% and 100& ETOH. Then on to Xylene x2 for 20-30
minutes at Room temp and vacuum and pressure in the #2 station. Paraffin -=20
infiltration is real important for good sectioning - I used all 4 paraffins=20
at 60 degrees C - 30 min. in the first one and an hour in the remaining
stations with vacuum and pressure. I embedded the critters - about 30-50
per block in Paraplast.
The sections were OK - some knife marks but for the most part the sections=20
were very good. I cut 5 micron sections on non-chilled blocks with minimal=20
soaking in RT water for in-situ hybridization.
IF THERE IS ANYBODY OUT THERE PROCESSING AND SECTIONING INSECTS WHO MAY
HAVE SOME BETTER METHODS PLEASE SHARE THEM!!!
At 11:01 AM 6/7/2006 +0200, Dr. Elke Genersch wrote:
>For in situ hybridization we need to obtain sections from the heads of
>bees (not just brain!) and from whole mites. Has anyone experience with
>fixation, embedding and sectioning of such material ?
>Elke Genersch, PhD
>Elke Genersch, Ph.D.
>Institute for Bee Research
>D - 16540 Hohen Neuendorf
>Tel.: +49 - (0)3303 - 293833
>Fax: +49 - (0)3303 - 293840
>Associated Member of the Zentrum für Infektionsbiologie und Immunität =3D ZIBI
>Histonet mailing list
: Andrea Grantham, HT(ASCP) Dept. of Cell Biology & Anatomy :
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