[Histonet] TEM and oysters

[Monfils, Paul]

(1)  For most types of antigens, not likely. Glutaraldehyde crosslinks
proteins so aggressively that most antigenicity is greatly reduced or
completely destroyed. If immunolabeling is the objective before the fact,
the tissue should be fixed very conservatively, for example 0.25%
glutaraldehyde for a half hour to one hour. Following normal glutaraldehyde
fixation with osmium introduces additional problems, as osmium greatly
denatures proteins, even those crosslinked by glutaraldehyde, and actually
dissolves some pre-fixed proteins out of the tissue by breaking the
glutaraldehyde bonds.  The only successful attempt at immunolabeling
glut/osmium fixed tissue that I can recall was a paper written by David
Knibbs around 1992-1994 in which he described a method for immunolabeling
(using colloidal gold-labeled antibodies) various kinds of intermediate
filaments.  I believe the abstract of that paper would be in the proceedings
of the American Society of Cell Biology.

(2)  You cannot "decalcify" mollusc shells in the same sense that you can
decalify bone because bone consists of a solid tissue matrix infused with
calcium salts, while a molluscan shell is composed almost completely of
calcium salts deposited by the animal's mantle, with only intermittent
traces of protein.  So, if you expose the shell to decalcifying agents, the
shell will dissolve completely.  If the loss of the shell is not a problem,
then it can be dissolved away much more rapidly than a bone of equivalent
size can be decalcified, since the bone matrix presents a barrier to the
penetration of the decalcifying solution. When I taught chemistry years ago,
I did a demo by filling a 1000 ml glass cylinder with 10% hydrochloric acid
and dropping in a clam shell.  A dense cloud of bubbles would immediately
erupt, causing the liquid to "boil" as the calcium carbonate of the shell
was transformed into carbon dioxide, and the shell would be completely gone
in about two minutes. So, theoretically any decalcifying method could be
used to remove the shell from a molluscan specimen. If you consider acid
methods too harsh, I would recommend a chelation method such as disodium
EDTA.  I never tried this on a mollusc shell, but I see no reason why it
wouldn't work. You'll have to experiment to find an optimum exposure time,
but again it should be much shorter than the time required for decalcifying
bone.

> ----------
> From: 	histonet-bounces <@t> lists.utsouthwestern.edu on behalf of
> Anne-Sophie MARTINEZ
> Sent: 	Monday, March 20, 2006 7:55 AM
> To: 	histonet <@t> lists.utsouthwestern.edu
> Subject: 	[Histonet] TEM and oysters
> 
> Hi everybody. I have 2 questions concerning TEM.
> (1) I have fixed gonads of adult oysters for TEM with glutaraldehyde, 
> post-fixed with osmium and done the embedding in Epon. Is there any way 
> I can you do immunocytochemistry with antibodies on these samples?
> (2) How can I decalcify the shelves of larvae and juveniles oysters for 
> TEM without damaging the tissue?
> Could anyone help me please? Thanks for your suggestions.
> Anne-Sophie
> 
> 
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