[Histonet] Crumbling frozen sections

[Mike Pence]

The difference you have is snap frozen tissue vs. tissue frozen by dry
ice and isopentane freezing.  You are more than likely getting freeze
artifact from the tissue setting around in a closed tube (moisture
build-up and ice crystal formation when frozen with iso.)  Try handling
your colorectal tissues in the same manner and see what happens.

Mike

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Martin
S.
Sent: Thursday, May 10, 2007 11:25 AM
To: Gayle Callis
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Crumbling frozen sections


 
I have tried a range of temps from -30oC to -10 (I went up in 5oC
intervals). For the colorectal tumour that was snap frozen cutting at
-20oC was good its just these new ones that I'm having a problem with.

The tissues are put in a plastic tube without anything else - just a
blob of tissue. I'm not sure how long the tissue sits like this before I
get it - the surgeon isnt very forthcoming with info!

I have been using APES coated slides (which I coat myself 5% APES in
Acetone, 5min, wash dH2O x3, dried in oven) and have done one test with
VWR Superfrost charged slides (no difference).

Thanks
Sonya


-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: 10 May 2007 15:55
To: Martin S.
Subject: Re: [Histonet] Crumbling frozen sections

Have you played with cryostat temperature to improve sectioning? It may
be too cold (you did not say what temp you cut at?).  Are the human
tissues 
coming to you fresh or have they been dunked into formalin?   Do they
put 
this on saline moistened guaze to keep it from drying out?

Also, what slides are you putting the tissues on, plus charge?

So many questions?

At 08:19 AM 5/10/2007, you wrote:
>Hi All,
>
>I have been doing a lot of frozen sectioning of mouse tissues. I remove

>the organ (spleen, lymph nodes, liver etc) from the mouse immerse it in

>OCT in a foil mould and then place in a bath of isopentane on dry ice. 
>This has been working really well and I have been getting good
sections.
>
>I am now looking at some human tissue (colo-rectal and liver tumours).
>I receive a small piece of tissue from the surgeon which I have been 
>treating as for the mouse tissue. However I am finding it very hard  to

>get good sections. The tissue seems very flaky and crumbly and either
>disintegrates on cutting or if I do get what looks like an ok section 
>by the time I have gone through the staining procedure it has 
>completely gone!
>
>I am not sure how long it takes between the tissue being removed and me

>getting the sample - maybe 30min - could this be the cause?
>
>I have some old colo-rectal tumours that were snap frozen in liquid
>nitrogen and they seem much better - I think I'll try doing this from 
>now on but just wondering if anyone has any insight into why I'm having

>such problems.
>
>Thanks!
>
>Sonya
>
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610








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