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This is forwarded by a colleague who remembered some more than I. Hope it
helps,
Fred Monson
-----Original Message-----
From: J. A. Kiernan [mailto:[log in to unmask]]
Sent: Monday, April 14, 2003 1:13 AM
To: Monson, Frederick C.
Subject: Re: formaldehyde fixation reversible?
Dear Fred, Feel free to forward this to the confolkers and
anyone else who might be interested.
The reversibility of formaldehyde cross-linking has been
known for a long time and receives attention in Pearse's
Histochemistry Vol 1 (and not just in latest edition, which
was 1980 for Vol 1; also in the 1968 3rd edn). Pearse gives
references. More recent studies by K.G.Helander are based
on binding and un-binding of radiolabelled formaldehyde.
They provide quantitative data but cannot distinguish bound
formaldehyde involved in cross links (N-CH2-N) from that
bound as N-CH2OH (to only one amino or peptide nitrogen).
Formaldehyde can bind similarly to oxygen (serine or
threonine -OH) and sulphur (cysteine). Cross-linking occurs
only at short range (the distance -CH2- between nitrogen (or
O or S) atoms in the tissue). For refs, see Pearse.
Removal of bound formaldehyde, whether involved in
cross-linking or not, requires hydrolytic cleavage of
the bond between a C from formaldehyde and an N
(or an O or an S) of the tissue. Chemists break C-N
bonds by heating alkaline solutions. That would be
too fierce for sections mounted on slides. Alkalinity
removes sections from glass even at low temperatures.
Helander's studies were of fixation and washing in cold water
for a variety of times. He did not investigate the actions of
hot water or of buffers with different pHs. His methods could
be used to investigate how antigen retrieval works, but I have
not seen any published investigation. The major papers on
antigen retrieval are full of intelligent speculation (=informed
guesswork) about how it works. Experiments to test the theories
do not require great intelects or expensive equipment. The
answers could be obtained quite simply if funds were available
to pay a few technicians' salaries and buy the suppplies for
2 or 3 years. Granting agencies don't find this kind of
research worthy of support, even though it might greatly reduce
the costs of troubleshooting in routinely used diagnostic
immunohistochemical procedures.
Here are the Helander refs.
Helander KG (1994) Kinetic studies of formaldehyde binding in
tissue. Biotech. Histochem. 69: 177-179.
Helander KG (1999) Formaldehyde binding in brain and kidney: A
kinetic study of fixation. J. Histotechnol. 22: 317-318.
--
-------------------------
John A. Kiernan MB, ChB, PhD, DSc
Professor, Dept of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
[log in to unmask]
http://publish.uwo.ca/~jkiernan/
_______________________________________________________________
> "Monson, Frederick C." wrote:
>
> But the purpose is the HCHO in the first place is two fold.
>
> First to kill - i.e., stop degenerative processes.
>
> Second to preserve.
>
> Somewhere between First and Second, one of the alternatives is
> another nucleophilic attack which forms a 'methylene' bridge -
> sigma bonds - from N to N (I believe) that are not as friable
> as the alternatives that Jim remembers better than I, and I had
> "Organic Mechanisms" as a course sometime! In any case,
> without trying to understand all the mechanics of the new
> "unfixing" solutions, I depend on what I understand from olden
> days when we used a base (1% KOH? - NOT too LONG now!) to
> reverse long-term, methylene-bridge, fixation.
>
> Time and temperature are relevant objects in the fixation
> program. I have 'fixed' the size of a distended urinary
> bladder by simply placing it full in cold, 4% HCHO, for 15
> min. After that, I have sampled it, cryoprotected it, frozen
> it, stored it at -80 for months, and finally sectioned it to
> stain with my Ab's, with no untoward results at all. I have
> never had to reverse fix for the Ag's in which I was
> interested.
>
> I have copied two colleagues with long experience who might be
> able to add to this thread, and correct me if I have misspoken.
>
> Cheers,
>
> Fred Monson
>
> Frederick C. Monson, PhD
> Center for Advanced Scientific Imaging
> Mail Drop: Geology
> West Chester University
> West Chester, PA, 19383
> http://darwin.wcupa.edu/casi/
> Phone/FAX: 610-738-0437
>
> -----Original Message-----
> From: James Sanzo [mailto:[log in to unmask]]
> Sent: Saturday, April 12, 2003 10:59 AM
> To: [log in to unmask]
> Subject: Re: formaldehyde fixation reversible?
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi
> Andi,
>
> It's an organic chem thing. Here's my shot at explaining it
> (Warning: this is pretty rusty stuff!):
>
> Formaldehyde, like every other aldehyde has a nucleophillic
> carbonyl as it's bond forming group. This means that aldehydes
> form bonds through "nucleophillic addition". In turn, this
> means that any molecule that has an electrophillic group can
> possibly be a bond former. The stability of bonds formed
> through nucleophillic addition depends on the particular
> electrophile the aldehyde combines with. Formaldehyde also
> readily reacts with water to form methylene glycol - a easily
> reversed reaction. The glycol plays a role in the fixation
> event, but we can ignore it for this discussion.
>
> As biologists, the prime reaction we depend on is protein
> fixation, which is addition of an aldehyde to an amine to
> produce an unstable "aminol". The aminol then usually
> dehydrates to an imine - which is more stable. However the
> aminol can revert back to the aldehyde + amine under the right
> conditions ("wrong" conditions as we would normally wish to see
> it) - through mass action, for example. The imine can also
> revert back to free aldehyde + amine by hydrolyzation.
>
> How much washing is too much? The answer depends on:
> - how well fixed the tissue was to begin with
> - quantities of other substances in the wash that may catalyze
> the forward reaction or reverse reaction (certain acids or
> bases)
> - the overall pH of the wash solution
> - temperature of the wash
> - time
>
> I think there's still a lot of confusion about formaldehyde
> fixation mechanisms. This is reflected in the literature and in
> products being sold for antigen retrieval: some are citrate
> based and acidic (~pH 3), some are EDTA based and basic (pH 8 -
> 9). * Perhaps someone with less rusty knowledge of the organic
> chem involved will pipe in here??
>
> Refs:
> Many books on immunohistochemistry and the like mention this
> phenomena. Gareth Griffiths book "Fine Structure
> Immunocytochemistry"is one. I also recall seeing something in
> the Hyatt books on EM. You should also check papers on antigen
> retrieval like: Shi, S.S, et al. Antigen retrieval techniques:
> current perspectives. 2001. J. Histochemistry & Cytochemistry.
> 49/8:931-937.
>
> Happy washing,
> Jim
>
> At 11:20 PM 4/11/03 -0500, you wrote:
>
> > Search the CONFOCAL archive at
> > http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> >
> > Dear Jim,
> >
> > I have not heard about the reversibility of a
> > formaldehyde fixation before. Do you have literature
> > about this unfixing effect? From your experience, how
> > much washing would be too much?
> > Andi
> > Michal
> >
> > The quality (or lack of) in a frozen, stored preparation
> > depends on whether the microstructure is disrupted by
> > ice or not. If the cells are permeabilized and stored in
> > a media that avoids damage from ice crystal formation,
> > the quality might not be too bad. Storing beneath the
> > surface of a media (or in a frozen block) will also
> > protect the cells from being dried out. Many
> > cryoprotectant formulations are available for frozen
> > sectioning which will probably work fine for your needs.
> > There is usually sucrose and/or a glycerin component
> > that needs to be infiltrated. PBS made with 30% sucrose
> > would probably be fine. Thatıs about it.
> >
> > However, remember that formaldehyde fixation is
> > reversible, and stored ³highly washed² cells will be at
> > the greatest risk of unfixing which most likely will
> > compromise the structure and turn it into glop.
> >
> > Good luck,
> >
> > Jim
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