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Though there are substantial differences between the following method, it
nonetheless demonstrates something all users of HCHO and fluorescence
microscopes should know.
Fuxe, K. & Jonsson, G. 1973 The histochemical fluorescence method for the
demonstration of catecholamines. J. Histochem. Cytochem. 21,293-311.
Regards,
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: Mario M. Moronne [mailto:[log in to unmask]]
Sent: Tuesday, April 15, 2003 7:35 PM
To: [log in to unmask]
Subject: Re: formaldehyde fixation reversible?
Search the CONFOCAL archive at
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Folks,
I know that someone will write a succinct summation of the pluses and
minuses of using formaldehyde for fixation and Fred's list is getting
there. But, a) I am not going to write it and b) I still want to add
a couple of things.
Of every fixative I have tried, which includes the crosslinking
polymerizable aldehydes, and the denaturing and dehydrating solvents,
formaldehyde provides the lowest autofluorescent background generally
eliminating the need for any borohydride reduction. However, unless
my organic chemistry is "wack" (sorry I just saw the EMIMEM movie),
the borohydride should prevent the reversible binding of formaldehyde
to the protein. Also, as a rule of thumb, and someone please correct
me if I am wrong, formaldehyde as a fixative being such a small
molecule is less likely to damage or destroy epitopes for antibodies,
and because it is not bifunctional in the manner of glutaraldehyde,
it is much less capable of forming the long conjugated (and quite
fluorescent) polymers that is the basis for glutaraldehyde fixation.
Although, I have not tested this quantitatively, the normal cell
autofluorescence of mammalian cells in culture appears hardly
different than the autofluorescence of cells that are formaldehyde
fixed (e.g., 488 nm ex, 520 em).
This is hardly the case for glutaraldehyde (Glut.). Although it is
wonderfully stable and was a great advancement for EM, the complex
polymers that grow from the protein-Glut. Schiff bases are very
fluorescent. Colleagues of mine have even used this property as a
general stain for highlighting zymogen granules that have very high
concentrations of protein and thus bind a lot of Glut. Using
borohydride reduction does reduce the Schiff's base stabilizing the
link (not that it really matters because of the glue that is
essentially formed using Glut.) and further borohydride does a modest
job of quenching the Glut. related fluorescence. However, I have
never seen this "autofluorescence" completely eliminated even after a
number of borohydride treatments.
It is probably in the archives someplace, but my technician of some
years ago and I spent two months doing nothing but different
labeling techniques of microtubules. We ultimately found that
glutaraldehyde never could compete for producing low autofluorescent
backgrounds compared to formaldehyde. Our protocol also started by
treating our 3T3 cells at 37 deg. with low concentrations of taxol
first to promote tubule polymerization then followed by DSP
pretreatment to provide a little crosslinking and stabilization of
the microtubules. Then we used many different combinations of
formaldehyde (with and without pH shifting), glutaraldehyde (plus or
minus borohydride reduction, formaldehyde plus Glut., denaturing
solvent protocols such as cold methanol, ethanol, acetone sometimes
with formaldehyde pretreatment and three different anti beta-tubulin
antibodies, only one of which worked very well (an ICN primary
anti-beta, very nice but that was 6-7 years ago.)
We came to the conclusion that adding glutaraldehyde always incurred
the risk of severe autofluorescence that could only be reduced by
maybe a factor of three with borohydride - a significant reduction
but still a great deal of background fluorescence remained compared
with results using formaldehyde as the fixative.
I have additional related concerns regarding Glut as a fixative. From
x-ray microscopy imaging where image contrast is generated primarily
by carbon k-edge absorption (~284 eV) in aqueous media, the amount of
fixative actually bound to cells is clearly non-trivial such that one
must wonder how much ultrastructural changes are produced or even
created artifactually by the glutaraldehyde. The amount of bound
fixative can actually be calculated using x-ray absorption. This was
very apparent when I first tried imaging red blood cell ghosts at the
National Synchrotron Light Source (NSLS, Beamline X-1A) at the
Brookhaven lab. Without Glut. it was very difficult to visualize the
red cell ghosts but once treated with Glut. the contrast increased by
a factor of about three making the RBC membrane very distinct. This
suggested extensive binding of the fixative such that the fixed
membrane mass may have come close to tripling. This particular study
was never concluded but to my mind it strongly suggested that
glutaraldehyde adds possibly many layers of fluorescent plastic like
polymer.
So that is my 2 cents worth. Borohydride reduces Schiff's base which
should mostly eliminate reversibility for any aldehyde-amino adduct.
Glutaraldehyde produces drastic changes at the molecular level
compared to formaldehyde and adds greatly to autofluorescence that is
not easy to remove.
Regards All and thanks Fred for all your time and effort of late,
Mario
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>Fast penetration --> rapid killing
>Smooooooooth texture in finished product.
>Secret mechanisms of REAL action that keep everyone coming back.
>Reasons for using HCHO.
>
>Habit
>Protocols for 100+ years
>Easy to use and relatively safe to keep as paraformaldehyde
>VERY wide range of concentrations that work (2-~40%)
>Works as gas (dry) and in aqueous solution.
>Habit
>Progressive fixative - some of the chemistry is fairly clear
>Reversibility is relevant to IHC-Ag recovery, but not to much else. But, I
>repeat, the progressive addition stage of HCHO fixation does not occur in
>significant degree, according to some, until 12+ hours after exposure
>begins, and the earlier process might have more to do with the reductive
and
>toxic (whatever that means mechanistically, I do not know) capacities of
>HCHO than on the question of addition and/or subtraction.
>
>[Biological systems are well-balanced thermodynamic systems, but HCHO is
>most likely so popular, because it is effective in rapidly diffusing
>throughout the system, poisoning both anabolic and catabolic systems, and
>SLOWLY thereafter reacting directly with adjacent macromolecules to provide
>crosslinks that progressively preserve the structural relationships of
those
>cellular and tissue components.]
>
>[In urology it is common, for some chronic irritative bladder dysfunctions,
>to instill 50% DMSO (for a little while under anesthesia-I think) as a
>palliative. One of the early papers, while reporting on the therapeutic
>efficacy of various concentrations of DMSO, notes that 50-100% DMSO is also
>a very good histologic fixative for bladder epithelium.]
>
>Summary: Rapid penetration, killing and halting degenerative changes are
>not mutually exclusive criteria for a good chemical fixative. It is
>interesting that, except in pathology and electron microscopy, HCHO has
>almost replaced all of the old, heavy metal fixative formulations.
>
>Cheers,
>
>Fred Monson
>
>
>-----Original Message-----
>From: Guy Cox [mailto:[log in to unmask]]
>Sent: Tuesday, April 15, 2003 4:04 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
>
>Scott Snyder wrote:
>
>>What I have often wondered is why formaldehyde is so popular given its
>>variability >from batch to batch, reversibility, pH dependence, and
>>potential for forming unwanted fluorescent adducts.
>
>Nobody in this field uses bottled formaldehyde, precisely because of
>this variability. We prepare formaldehyde freshly from paraformaldehyde
>and hence obtain a fresh and uniform product. When permanent
>crosslinking is required glutaraldehyde is the fixative of choice -
>the great advantage of formaldehyde is its ability to provide
>a milder and less permanent stabilisation. If you could offer
>rivals to these two which didn't induce fluorescence I'm sure a lot
>of people would listen!
>
>About the only other protein cross-linking agent I know that has
>achieved any wide use is MBS (m-maleimidobenzoyl N-hydroxysuccinimide
>ester) introduced by Sonobe & Shibaoka to stabilise actin (Protoplasma
>148, 80-86 (1989)). A similar (and I can't face typing it) substance
>has been used to stabilise microtubules. I've certainly used MBS
>in the past and I reckon it works, but I'm unclear why one needs a
>different reagent for a different protein (tubulin). Maybe that's
>why the aldehydes remain popular?
>
> Guy
>
>Assoc. Prof. Guy Cox, ooOOOOOOoo
>E.M. Unit, F09 # oOOOO | | OOOOo #
>University of Sydney ### OOO| | | | | |OOO ###
>NSW 2006, Australia ### OOO | | | | | | OOO ###
>Ph: 02 9351 3176 ### OO | | | | | | | | OO ###
>Fax: 02 9351 7682 ##### | | | | | | | | #####
> ==#####============================#####==
>http://get.to/gcc ##### #####
>http://guycox.cjb.net ~~#####~~~~~~~~~~~~~~~~~~~~~~~~~~~~#####~~
--
_________________________________________________________________
Mario M. Moronne, Ph.D.
NanoMed Technologies
ph (510) 528-2400
FAX (510) 528-8076
1561 Posen Ave
Berkeley, CA
94706
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