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

[Margaret Blount]

Storing your samples overnight in 70% ethanol after fixation won't harm your samples, but they should be adequately fixed prior to this. I do not like xylene for mouse tissues, it makes them too brittle. Instead I used histoclear 11 from National Diagnostics. Histoclear 11 is less expensive than the original histoclear, but performs well in my experience. I found that for most of the tissues I processed (I never tried diaphragm) a shorter schedule worked well and in fact the process you describe may be too long for some tissues. The Society of histotechnologists produces a booklet with suggested processing schedules for a wide range of animals and tissues, I found this invaluable in designing my protocols. My process for mouse tissues took aroung 6 hours using a processing machine equipped with vacuum on all stations. Unfortunately as I have retired I don't have access to my SOP's any more. However, if you obtain the booklet from the Society, you will be able to devise a good protocol. At the end of the day, if your tissues section and stain well, then your process is satisfactory. 
It's always helpful to compare your sections with other people's if you can then you have a benchmark of quality. This can be difficult and frustrating in a small research lab. 
 
You may find that postfixing your liver samples in formol alcohol (90ml absolute ethanol to 10ml 37% formaldehyde or use Pen fix from thermo shandon) will improve the morphology. I used to do this for a couple of hours; you may need to experiment, especially if you are doing immunohistochemistry which is presumably why you are using paraformaldehyde. 

 

Good luck

 

Margaret


________________________________

From: histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Itai Moshe
Sent: Thu 07/07/2011 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
Doe
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