[Histonet] sectioning bee's heads and mites

Dr. Elke Genersch elke.genersch <@t> rz.hu-berlin.de
Wed Jun 7 12:15:46 CDT 2006

Mr. Grantham,

unfortunately, I can't do paraffin sections. I have to use historesin as 
embedding media since the sectioning is done in another lab where only this 
embedding method is used (and liked). We found a paper (Brazil et al., 
2003; comparison of different fixation and infiltration techniques) where 
sand fly specimens were fixed in Bouin/Carnoy and embedded in historesin 
with OK results - so they wrote. It did not work out as described with our 
bee heads and mites. If you could scan and pdf the pages from the "Manual 
of bacis techniques in insect histology" you refer to - or any other pages 
relating to my problem - it would be great. We don't have the book in the 
Is Penfix compatible with historesin?

Elke Genersch

At 08:53 07.06.2006 -0700, Andrea Grantham wrote:
>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.
>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
>>Histonet mailing list
>>Histonet <@t> lists.utsouthwestern.edu
>: 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)       :
>           http://www.cba.arizona.edu/histology-lab.html

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