[Histonet] Advice.

[Maria Mejia]

Ian,

Hmmm...well, some years back I was working on a butterfly heads project 
for a brain
cell count study. Never worked on insects before, so it didn't take long 
before I noticed
the head of the butterfly is covered with chitin layers and I had to 
section the entire head.
Here's what I did. I got beautiful paraffin sections stained w/Nissl 
staining.

With agitation:
 > fixed cut head in Carnoy's fixative - overnight @ 4C (there are many 
formulas in
the literature. Possible could also use glutaraldehyde containing 
fixative - sure these
would be fine for LM too)
 > dehydrate a bit more with ethanol alcohol 2x - 30' each
and then
 > soak (tissue) in a mixture of equal parts of chloral hydrate:phenol 
to soften the entire
chitin.
*Chloral hydrate:phenol made by weighing equal parts & then gently 
warming until
molten. Once molten can store for several weeks. Sections cut beautiful.

Tissues were left in this mixture for a minimum of 2 hours to overnight 
(perhaps even up
to a couple days).
 > clear in chloroform 3x - over 2 hours to overnight (I did 1 hour each).
 > place in cedarwood oil - 2 days (change oil 3x during period).
 > then do routine paraffin.
*When sectioning, I remember adding a bit of glycerin in the ice water 
to soak the blocks a
bit.

Ian, I hope this helps.

Maria Bartola Mejia
Smith-Kettewell Eye Research Institute
San Francisco, CA 94115
Email: maria <@t> ski.org
Phone: (415)-345-2185


Ian Montgomery wrote:

>         With cuts in staff I've been asked if I can teach the 
> histology portion of a zoology class. Problem, one of the tissue I'll 
> be using is insect abdomen with its chitin exoskeleton. Any hints and 
> tips regarding this type of material, processing, embedding, 
> sectioning, that sort of thing. Or, is it Mollifix time again?
>
> Dr. Ian Montgomery,
> Histotechnology,
> IBLS Support Services,
> Graham Kerr Building,
> Institute of Biomedical & Life Sciences,
> University of Glasgow,
> Glasgow,
> G12 8QQ.
> Tel: 0141 339 8855
> Office: 4652
> Lab: 6644.
> Pager: 07623 975451
> e-mail: ian.montgomery <@t> bio.gla.ac.uk 
>
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