[Histonet] sectioning bee's heads and mites

[Andrea Grantham]

Dr. Genersch,
 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 
easier when faced with sectioning these critters. It involves using Butanol 
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 
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 
completed about the same time I found the book.
Also - another method that works OK: the investigator who brought the last 
insect project to my lab fixed whiteflies in Penfix (Richard Allen 
Scientific). He removed the wings and legs and continued fixing for 36 hrs. 
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 
the dissecting microscope pulling off their little legs and wings? Sounds 
evil!
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 - 
infiltration is real important for good sectioning - I used all 4 paraffins 
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 
were very good. I cut 5 micron sections on non-chilled blocks with minimal 
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!!!
Good luck!
Andi Grantham


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.
>Vice Director
>Institute for Bee Research
>Friedrich-Engels-Straße 32
>D - 16540 Hohen Neuendorf
>
>Tel.: +49 - (0)3303 - 293833
>Fax: +49 - (0)3303 - 293840
>e-mail: elke.genersch <@t> rz.hu-berlin.de
>homepage: www.honigbiene.de
>
>Associated Member of the Zentrum für Infektionsbiologie und Immunität = ZIBI
>www.biologie.hu-berlin.de/~ZIBI/
>
>
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.....................................................................
: Andrea Grantham, HT(ASCP)     Dept. of Cell Biology & Anatomy     :
: Sr. Research Specialist       University of Arizona               :
: (office:  AHSC 4212)          P.O. Box 245044                     :
: (voice:  520-626-4415)        Tucson, AZ  85724-5044    USA       :
: (FAX:  520-626-2097)          (email:  algranth <@t> u.arizona.edu)       :
:...................................................................:
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