[Histonet] frozen cryoprotected tissue

[Melissa Gonzalez]

Dear Phil and Patsy, 
Unfortunately, I do not have an answer for your question, and would be curious myself to see if anyone has a solution to this problem. 

While I have you guys on the fixed, sucrose cryoprotection and then freezing method of processing tissues, I have some questions regarding the process. 
1. First, how do you freeze the tissue when it is ready (after sucrose)? 
I have known people to freeze it directly in OCT in the cryostat or -20C, or also a quick freeze as you would fresh frozen tissue in a dry-ice isopentane bath. I myself prefer the slower freeze, as the tissues always seem to cut much better, and have never seen any compromise in morphology/staining. When the tissues are already fixed and cryoprotected, does the temp really matter?
2. I have however, noticed that regardless of the freezing temp, the H&E's appear strange. The cells are usually separated and the nuclear detail is very poor. Even though the H&E process is the same we would use for fresh frozens. So the fresh frozen slides actually turn out better than the fixed, cryoprotected samples. 
(Our processing procedure is as follows: after resection, post-fix tissue in 4% NB paraformaldehyde 3 hrs RT, followed by 30% sucrose immersion overnight at +4C or until tissue sinks, followed by (slow/quick) freezing in OCT. 

Any comments?

Thanks
Melissa

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Hello all,

   I was wondering if anyone has had any problems with tissues that were 
quick frozen before being being fixed and cryoprotected?  We usually obtain 
our tissues fresh, fix in some sort of paraformaldehyde solution (Zamboni's, 
Lanas), cryoprotect in sucrose solution for around 1 week, and then quick 
freeze.  However, for our positive controls, we are only able to obtain 
quick frozen tissues that haven't yet been fixed.  I suppose that this might 
affect the achitecture a bit, but does anyone have any knowledge of whether 
immunohistochemistry might be affected?  We are looking for nerve 
innervation, and will be obtaining muscle and skin for these purposes....

-Phil




Message: 14
Date: Tue, 28 Jun 2005 09:11:53 -0600
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
Subject: RE: [Histonet] post-fixing quick frozen tissues for IHC
To: "'phil tsai'" <fillsigh <@t> hotmail.com>,
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200506281511.j5SFBikM012810 <@t> chip.viawest.net>
Content-Type: text/plain;	charset="us-ascii"

I have the same issues, I get tissue quick frozen without any OCT or
surcrose cryoprotectant from the molecular biologists all the time,
sometimes the tissue is not even quick frozen it is just taken fresh and
stuck in a tube in the -80dc freezer and the morphology is terrible.   There
was a technique a year ago in Histologic about thawing and refreezing poorly
prepared samples, I have tried this with some improvement but it is never as
good as doing it right in the first place.  I would be interested in others
approach to this issue as I don't see an end to getting samples not properly
prepared for frozen sectioning.
Patsy