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

From:"Dr. Elke Genersch"

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=20
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=20
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=20
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=20
> 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=20
> 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.=20
> 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=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. Rinsed in 70% ETOH and removed any remaining wings and legs he missed=20
>the first time. A tedious task for something as small as whiteflies! Glad=20
>he was doing it!!! Can you just picture this guy sitting there looking 
>through the dissecting microscope pulling off their little legs and wings?=20
>Sounds evil!
>I processed on a MVP tissue processor, 15 min in each station starting at=20
>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 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 = 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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