[Histonet] re: fixation question for IHC (Jan Berry)

John Kiernan jkiernan <@t> uwo.ca
Sat Jan 29 23:02:24 CST 2011

I agree with Jean-Martin Lapointe. The small amount of methanol in a neutral buffered fixative made from formalin could have no fixative (coagulant) action. You need something approaching gin or vodka (40%v/v) to get ethanol  to coagulate proteins and kill bacteria. Museums suggest vodka as a "spirit" preservative for specimens sent in by amateur naturalists. 
Very old formalin contains a little less formaldehyde, a little more methanol, and also some formic acid, but the acidity is dealt with when the pH of a neutral fixative is adjusted. 
Formalin stored in a cold place usually contains less than the 37%(w/w) or 40%(w/v) of formaldehyde stated on the label. The missing formaldehyde is is in the white precipitate (which is paraformaldehyde) in the bottom of the bottle. 
Fortunately for all who use formaldehyde as a fixative, the concentration probably does not much matter within the range 1% to 40%.  I'm uncertain about the lower limit, but have encountered colleagues fixing animals brains in neat formalin for subsequent thick frozen sections to locate electrode placements. JF Walker's book "Formaldehyde" 3rd ed 1964, available as a reprint (Krieger 1975, ISBN 0882752189) cites many "preservative" uses of formaldehyde at concentrations lower than our customary 4%.
Pretty good structural preservation for electron microscopy is possible with an intelligently formulated solution made from formalin with optimal salt concentrations and pH. Look up Carson FL, Martin JH & Lynn JA (1973) Am. J. Clin. Path. 59: 365-373.
John Kiernan
Anatomy, UWO
London, Canada
= = =
----- Original Message -----
From: Jean-Martin Lapointe <jm.lapointe <@t> accellab.com>
Date: Friday, January 28, 2011 15:18
Subject: [Histonet] re: fixation question for IHC (Jan Berry)
To: histonet <@t> lists.utsouthwestern.edu

> I think it is a very small minority of markers that would be 
> sensitive to the methanol residues present  in formalin. So 
> it depends on what you plan on staining. For general purposes, I 
> would always go with formalin, unless there is a proven need for 
> formaldehyde.
> __________________________________
> Jean-Martin Lapointe, DMV, MS, dACVP
> Vice-President, Pathologie
> AccelLAB Inc
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> jm.lapointe <@t> accellab.com
> ------------------------------
> Message: 3
> Date: Fri, 28 Jan 2011 13:15:19 -0500
> From: Jan Berry <jeberry <@t> umich.edu>
> Subject: [Histonet] fixation question for IHC
> To: histonet <@t> lists.utsouthwestern.edu
> Message-ID: <1BA205BB-22B9-4167-A36D-01B7F3F04A0B <@t> umich.edu>
> Content-Type: text/plain; charset=us-ascii
> Is there a difference between using paraformaldehyde and neutral 
> buffered formalin when choosing a fixative for IHC?  I 
> would prefer to use formalin because of easier preparation, but 
> am willing to put in the extra time to make fresh 
> paraformaldehyde solution if there is a compelling reason.
> Jan Berry
> University of Michigan
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