Re: "xylene AND alcohol substitude" & "rabbit spleen in formaldehyde 37%"

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From:Yvan Lindekens <yvan.lindekens@skynet.be> (by way of histonet)
To:histonet <histonet@magicnet.net>
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hi all,

ETOH and xylene can be ommitted in the embedding process (paraffin) by the
use of 2-propanol. This calls for dehydration in 2-propanol at room
temperature, 2-propanol at 50C, 2-propanol-paraplast mixture (1/1 v/v) at
50C and finally, normal embedding in Paraplast.

I use this method all the time (histology and plant anatomy); It works very
well... I have tried it with several other paraffin's (Paraplast-plus,
"paraffin fur die histologie", Merck and even with a mixture of household
paraffin, mp. 52C  and purified biewax (95/5 w/w). The results were OK,
sometimes even better compared to the results obtained by "classical
techniques". Cutting artefacts are far less, due to the abcense of the
hardening effect of xylene (benzene, toluene)...
(sections 4-12m, RT= 16C, with manualy operated rotary microtome and
c-type knive: as an amateur, I don't believe in disposable blades: grinding
and polishing knives is part of the fun).

One drawback: dehydration trough 2-propanol is slower than dehydration
trough ETOH...

The method is described in Bck, P.: "Romeis' Mikroskopische Technik",
1989, Urban&Schwarzenberg, ISBN: 3-541-11227-1.

It's a modification of an old technique (Dietrich, 1929; Doxtader, 1948)
and I think (but I'm not sure) that even Bolles Lee mentioned it in one of
the first editions of "The Microtomist's vademecum"... (If my dear friend x
would be so kind to, finally, return my copy? Thanks).
___________________________________________________________
There was a messages about rabbit spleen, "par accident" fixed in
formaldehyde 37% (Mrs. Mary Kay Olson). Embarrasing (know it, been
there...).

Perhaps you could give the 2-propanol-paraplast technique a try instead of
a regular ETOH-xylene-parrafin run: tissue hardening will be less and
perhaps it will be possible to cut usable sections... Cutting on a sliding
microtome (instead of a rotary-) could help too...

Mr. Paul Millikin said in his response: "...spleens are VERY hard to fix
WELL... ...They tend to be so full of RBC that they behave like a thrombus
or an embolus... When simply thrown intact into an immersion fixative, the
outer, subcapsular 2-3 mm tends to fix, but the rest is usually rotten...
... We've been looking at rotten, autolyzed tissue for too long...
...Slicing (breadloafing) the specimen every 2-3 mm before immersion causes
considerable improvement, and should be a must for everyone interested in
spleens".

This is certainly true... I always slice spleen (and other hard to fix
tissues as lung etc...) in slices about 2-3 mm thick. This can easily be
done with a sharp razor, a long razor blade or a large dissecting knive (no
scalpel: the blade is too "short"...) and without using pressure.
Even then, adequate fixation isn't always guaranteed when using NBF... I
believe it's better to fix in an alcoholic Bouin formulation
(Duboscq-Brasil, Lang, Rossman...)(depending on the purpose of the
preparations).
I know that there are conflicting opinions regarding the fixation of spleen
in Bouin, but if I have to choose between techniques that "are not generaly
accepted" and making slides of "rotten, autolyzed tissue"...

Yvan Lindekens.




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