Re: [Histonet] sectioning bee's heads and mites

From: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=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.
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
>Associated Member of the Zentrum für Infektionsbiologie und Immunität =3D ZIBI
>Histonet mailing list

: 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:       :

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