AW: [Histonet] fixation question

[Gudrun Lang]

Hi!
In my opinion 24 hours should be the minimal duration for fixation in 4%
buffered formaldehyde. The shorter the time the more differences may occur.
It is important to prepare the specimens in the same way at the grossing -
means same size of tissue in the cassette or at least minimal size of 3 mm
diameter.  24 hours of a whole organ in formalin is not the same as 24 hours
of a small tissue-block.
Look at the article of Cecil Fox about formaldehyde. He says that shrinkage
is not due to formalin, but more caused by the processing, when lipids and
water are removed.
The better the crosslinking, the less sensitive is the tissue to shrinkage
or swelling.
Immunhisto can be adopted to longer fixation, but too short fixation may
give impact on the morphology, that can't be recovered.

Shrinkage during processing can be intensified by very long times in
absolute ethanol and xylen. Let you inform about the processing protocol of
the company. The protocol should fit to your specimens.
On the other side inform the company about your fixation times. They may
prolong the antigen retrieval for better results.
My advice without knowledge of your special experiment is a fixationtime of
24-72 hours. 

Bye
Gudrun Lang


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Von: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von
ncosenza <@t> siumed.edu
Gesendet: Samstag, 19. Oktober 2013 16:51
An: histonet <@t> lists.utsouthwestern.edu
Betreff: [Histonet] fixation question

Hello Histonetters:

Our lab has a resident wanting to start a project involving drug effect on
tumor size.  Since he is wanting to compare tumor size at certain time
points after drug treatment (and do IHC staining intensity), he wonders how
much affect varying fixation times will have on the tissue.  We are not
doing the processing or embedding ourselves so we can't control for such
variation.

Is this a legitimate concern? If one piece of tissue sits in fix 24 hours
before processing/embedding, will the morphology be drastically different
than a second piece that sits longer (or shorter) than 24 hours?

Is so, would fixing the tissue ourselves for a set time and then putting in
some sort of buffer or ethanol before we ship the tissue to the company who
embeds it make a difference? The resident is really trying to minimize
swelling or excess shrinkage of the tissue.

Thanks!


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