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Couldn't agree more.  I tried a few cross-linking agents some years ago
when I was having trouble preserving cell structure, and some preserve
ultrastructure beautifully, at least at the light level.  A characteristic
of glutaraldehyde or formaldehyde fixation in plant cells is that the more
delicate endomembrane systems tend to vesiculate - these crosslinkers
preserved this perfectly.  At the time I needed to do antibody staining
after fixation, and the tissue was so well-fixed it could not be labelled.
I figured out a way to do the labelling with formaldehyde so have never
followed this up, but it would be worthwhile, esp. with all the new
water-soluble cross-linkers.  Could be very worthwhile for fixing
GFP-tagged cell components.

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal     Your  analysis
>is correct, based on my knowledge of the chemistry involved.  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.  Chemistry has given us a wealth of
>protein  crosslinking reagents many of which have been used successfully
>in fixation and  yet are largely ignored.  Compounds such as DSP, for
>example, can form  stable amide bonds with proteins which are essentially
>irreversible.  I  think this may be a case of many people having
>experience with something that  usually works well enough and being too
>busy to spend any time  trying something which may be better.
>
>   -----Original Message-----
>From: Confocal Microscopy List    [mailto:[log in to unmask]]On
>Behalf Of James    Sanzo
>Sent: Saturday, April 12, 2003 9:59 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. That's about it.
>
>
>However, remember that formaldehyde fixation is reversible, and stored
>       "highly washed" cells will be at the greatest risk of unfixing -
>which        most likely will compromise the structure and turn it into
>glop.
>
>
>Good luck,
>
>
>Jim