Re: osmium for autofluorescence.
|From:||"J. A. Kiernan" <firstname.lastname@example.org>|
On Fri, 6 Jul 2001, Debra Dunlap wrote:
> I am currently using osmium tetroxide to reduce autofluorescence ...
> ... fluorescent probes ... osmium seems to be quenching the
> fluorescent probe signal as well.
I hope I'm wrong and it's just a misunderstanding or words,
but if the "fluorescent probe" is something applied to
living or otherwise fixed cells/tissue, then osmium will
quench it. OsO4 is useful for quenching autofluorescence
_before_ applying something fluorescent to a preparation.
> ... I think Dr. Kiernan's suggestion of rinsing overnight should
> help a lot. I have only been rinsing for 30 min. What am I actually
> rinsing away? Is it free osmium tetroxide or am I hydrolyzing the
> cyclic osmic ester?
That's a really smart question Debra; it shows that you've
been doing your homework! I can't provide a very smart answer -
only partly informed guesswork. The osmium in a cyclic ester
is firmly bound to the lipid molecule. It won't be removed
(hydrolysis) by water but it can react with alcohol and end
up as insoluble OsO2 (black, if there's enough there to see).
The washing is to remove unreacted OsO4. Osmium is retained
in tissue at sites other than unsaturated bonds of lipids.
It slowly oxidizes all sorts of parts of proteins etc, with
the reduced Os ending up probably as insoluble OsO2 that is
not abundant enough to be black under the microscope, even
though the section may look grey to the unaided eye.
The suppression of fluorescence is supposedly due to the
large number of electrons that surround any atom with a
high atomic number. A section briefly racted with OsO4 and
then thoroughly washed contains enough residual osmium
atoms to quench immediately adjacent sources of
autofluorescence, such as molecules of lipofuscin,
flavins etc. The "background" seen in sections of many
tissues is also completely squashed by a few minutes in
an OsO4 solution..
A fluorochrome applied after quenching the autofluorescence
with osmium tetroxide typically will stand out in splendor
against a truly black background. This works wonderfully
with fluorescent methods for carbohydrates. BUT if your
fluorescent "probe" targets some part of a macromolecule
that is already close to an osmium atom, the fluorescence
of the bound probe could be quenched.
If you want some references to support these statements
about suppressing autofluorescence, I can provide them.
Please address any such request to Histonet at large and
not to me individually.
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London, Canada N6A 5C1
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