Re: Archived tissue viability when stored in 10% formalin

From:Frouwke Kuijpers

Are there reasons why you shouldn't store tissue in alc 70% after the normal
fixation procedure (IHC) as Tim Morken mentioned a few mails ago??

Frouwke Kuijpers

----- Original Message -----
From: "Gayle Callis" 
To: ; 
Sent: Friday, May 02, 2003 4:25 PM
Subject: Archived tissue viability when stored in 10% formalin

> Poor tissue is probably because the formalin has oxidized and pH is very
> acid. It might be worth a pH check to test pH.   Tissues stored for a long
> time should have the formalin changed regularly to maintain proper pH.
> has happened to us, but with bovine bone, the bone was actually decalfied
> after many years!!
> Here is a discussion I had with Dr. Fred Monson about formalin
>  He enlightened me on the chemical nature of formalin oxidation, a
> wonderful lesson in what happens to formalin.
> *****************************************************************
> The inquiry about old bones stored in formalin for 5 years.  Unless the pH
> of the formalin was maintained at pH 7, i.e. changed to maintain fresh
> you may encounter problems. Formalin tends to break down to formic acid
> time, the pH of "NBF" becomes acidic, and bone can also become
> decalcified. We had bovine bones stored for many years without changing
> NBF, and when trying to do undecalcified PMMA method, the bone literally
> twisted apart in the plastic block.  We should have stored the bone in 70%
> alcohol IF we wanted undecalcified bone.
> If NBF has been allowed to drop below 6.8 or lower, nuclear staining may
> also be damaged by this lower pH. You can try so see what happens, but be
> prepared for some changes.  Heavy duty cortical bone may withstand storage
> better than trabecular bone and soft tissues.  You might have to adjust
> your staining to have optimal results.
> Gayle Callis
> Fred Monson reply:
> I agree with every word, but would slightly amplify one, just FYI and for
> future reference.
> The single-carbon sequence is as follows:
> 1. CH4 methane,
> 2. CH3OH methanol,
> 3. CH2O methanal (or formaldehyde), and
> 4. CHOOH methanoic acid (or formic acid).
> There are some rare variations on this sequence, e.g., #3 can be a diol by
> having the =C pick up an H+ to give a second -OH.  This diol is often
> stipulated as the "active" form of formaldehyde.
> Each step (1-4) represents an oxidation, not a "breakdown".
> Frederick C. Monson, PhD
> Center for Advanced Scientific Imaging
> Mail to Geology
> West Chester University of Pennsylvania
> Schmucker II Science Center, Room SS024
> South Church Street and Rosedale Avenue
> West Chester, PA, 19383
> Phone:  610-738-0437
> eMail:
> Gayle Callis
> Research Histopathology Supervisor
> Veterinary Molecular Biology - Marsh Lab
> Montana State University - Bozeman
> S. 19th and Lincoln St
> Bozeman MT 59717-3610
> 406 994-6367 (lab with voice mail)
> 406 994-4303 (FAX)
> email:

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