query about methanol vs paraformaldehyde fixation

Tom Anderson via methods%40net.bio.net (by ucgatan from ucl.ac.uk)
Wed Sep 12 17:29:24 EST 2007


On Fri, 7 Sep 2007, [big5] Ĭ¨Øªâ wrote:

> Dear Dr. Schul,

You've emailed the Methods and Reagents mailing list, not Dr Schul.

> I found the following mail from google when I tried to find out what's
> the differences between methanol and paraformaldehye fixation. Is these
> messenger true?

It is.

Random things i can add to Dr Schul's comments:

- You get a better preservation (when using formaldehyde) by using a
friendly buffer (look up BRB80) and fixing your cells at 37 degrees (using
pre-warmed fixative and leaving the cells in the incubator to fix); cold
is one of the things that upset cells, and it can cause them to do funny
things before they get fixed. You see a significant difference in
microtubule staining between room-temperatureand warm fixations, for
instance, with the most dynamic microtubule tips being lost in an RT fix.

- Methanol-free formaldehyde is better than regular formaldehyde, as the
methanol can make proteins aggregate.

- Some antibodies only recognise their target in formaldehyde-fixed cells
(because methanol denatures the protein), and some only in methanol-fixed
cells (because formaldehyde chemically alters the protein, or because
methanol fixation denatures it and exposes the epitope). Most work in
both.

- If you want the preservation of a formaldehyde fixation, but the
immunoreactive properties of a methanol fix (if you have an antibody which
only works in methanol), you can fix in formaldehyde, then afterwards fix
again in methanol. This doesn't always work - if your antibody doesn't
work on formaldehyde-fixed cells because the fixation destroys the
epitope, postfixing in methanol won't help.

- If you want the preservation and/or immunoreactivity of a formaldehyde
fix, but want the extraction of soluble protein you get with a methanol
fix, you can do a pre-extraction: we use the same buffer we use for
fixation (without the formaldehyde!), plus 1 ug/ml phalloidin and 0.1%
Triton X100, warmed, for 30-60 seconds immediately before fixation. This
can be a really useful technique if you're looking at a protein where the
interesting molecules are stuck to something, but there's a lot of it
floating around too. I work on the cytoskeleton, so this is often the
case!

> Which articles can I use for reference, if I need to discuss with my
> boss?

I'm not aware of any papers discussing this explicitly; it's a matter of
art, something that's general knowledge in the community, rather than
something that's been studied. There may be papers on it, but they'll be
very, very old. There may be a paper specifically about the PCNA staining,
and if so, you should be able to find it on PubMed.

If you need to convince your boss about the difference, the simplest thing
might be to do formaldehyde and methanol fixes side by side and show him
the images!

tom

-- 
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264   (f) +44 (20) 76797805   (e) thomas.anderson from ucl.ac.uk



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