[Histonet] Leaving sample over night in ETOH 70% during fixation

[Rene J Buesa]

Bouin's and PFA are completely different in their action an results.
If you get good results with Bouin's use it and try to overcome your cutting difficulties.
There should be no differences using PFA or 37% formaldehyde. Methanol is only used to prevent formaldehyde polymerization and does not interfere with fixation. The problem is that you will need longer fixation times to obtain good results when using formaldehyde.René J.

--- On Thu, 7/7/11, Itai Moshe <itai.moshe <@t> mail.huji.ac.il> wrote:



From: Itai Moshe <itai.moshe <@t> mail.huji.ac.il>
Subject: Re: [Histonet] Leaving sample over night in ETOH 70% during fixation
To: "Edwards, Richard E." <ree3 <@t> leicester.ac.uk>, histonet <@t> lists.utsouthwestern.edu
Date: Thursday, July 7, 2011, 5:09 AM


I had a problem with liver fixation with PFA, most of the time I've got no
signal, so I've switched to Bouin's, and got good resalts, although it is
harder to cut bouin's sections with microtom.

Could it be because that I've switched from Paraformaldehyde powder to
formaldehyde solution (37%) (that contains methanol as a preservative) ?
I have one good PFA  powder experiment that I've done, and
some excellent old experiments that someone else in my lab did many years
ago, but i don't know how, all worked great with PFA fixation, but i could
not replicate the that.

I've tried to do a Formaldehyde 24Hr fixation R.T for the mouse liver
samples, but the results was catastrophic and even the DAPI didn't worked.

So i thought that maybe that because that the working samples were in 70%
ETOH over night.


2011/7/7 Edwards, Richard E. <ree3 <@t> leicester.ac.uk>

> Leaving  tissue overnight in 70% ethanol at 4C should have no detrimental
> effect n the tissues, why 4C overnight with Bouin's??
>
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:
> histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Itai Moshe
> Sent: 07 July 2011 09:22
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Leaving sample over night in ETOH 70% during fixation
>
> Dear All,
> Does leaving the samples (diaphragm, liver) over night in ethanol 70% at 4C
> during the fixation process, will be better for the tissue fixation, and
> does not harm the sample ?
> Does the fixation process should be done straight forward from the first
> step to the last one without any over night stops (except from the PFA,
> Bouin's step) ?
>
>
> My fixation protocol is like this:
> 1) immediately after killing the mouse i'm putting the sections in a
> fixation solution that is made from: 10ml formaldehyde 37%+5ml PBSx20+85ml
> DDW  - pH 7, Or bouin's solution over night at 4C.
> 2) ETOH 70%, ETOH 80%, ETOH 96%, ETOH 100% x2 - each for 1Hr at RT
> 3) Xylen x2 - each for 1Hr at RT.
> 4) Paraffin x3 - each at 60C for 1Hr.
>
> Thank you all very much in advance
>
> Itai M
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>


-- 
Itai Moshe
Mark Pines lab
Institute of Animal Sciences, Volcani Center.
Dept. of Animal Sciences, School of Veterinary Medicine, The Hebrew
University of Jerusalem
Israel
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