RE: Quenching autofluorescence
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|From:||"J. A. Kiernan" <email@example.com>|
|To:||Tamara Howard <firstname.lastname@example.org>|
On Fri, 28 Jul 2000, Tamara Howard wrote:
> There are quite a few things you can try. Someone has already
> suggested Cu sulfate and Sudan Black...I've had some success with treating
> the samples with a dilute (~0.5% aq.) Toluidine Blue stain (your local EM
> lab should have some stock on hand). I've also seen protocols for using
> Pontamine Sky Blue and some other dyes the same way...
> Depending on what is causing the autofluorescence, you may be able
> to kill or reduce it with a glycine (0.3M) wash or with a light Na
> borohydride treatment. There is another...with one of the ammonium salts,
> I think...but I'm drawing a blank. If you are interested I can organize an
> archeological expedition to my files and see if I can dig it up - although
> I'll bet that Dr. Kiernan knows it right off :)
Tamara has given a pretty good summary and can't add much to it other
than a few words about what "autofluorescence" comes from, because
that may determine the best way to try to quench it.
True native autofluorescence (as seen in living tissue cultures,
for example) is supposedly due in large part to substances like
flavins and porphyrins and (in plants) chlorophyll. These compounds
are generally extracted by solvents and aren't much of a problem
in fixed, dehydrated sections. They may persist (and be redistributed)
in frozen sections that have passed through various aqueous reagents.
I don't know of any published study, but there could well be several.
There's nearly always some "background" fluorescence (for unknown
reasons) in anything, especially with a broad-band blue excitation.
Narrow-band filters and lower wattage (50 rather than 200) mercury
lamps reduce this. If it isn't too bright, the background can be
helpful in seeing where you are in the section. Published fluorescence
micrographs are always printed so that the background is black and
only the significant fluorescence is visible. Lipofuscin is a native
autofluorescent material that persists even in paraffin sections.
It can be especially annoying in certain large neurons in the CNS.
Aldehyde fixatives react with amines and proteins to generate
fluorescent products. Glutaraldehyde is worse than formaldehyde.
I don't know about glyoxal-based fixatives, but hazard a guess
that they're somewhere in between. Glyoxylic acid (which is sort
of half glyoxal) was the best aldehyde reagent for demonstrating
amine fluorescence in the 1970s. The simplest way to stop
aldehyde-induced fluorescence is to use a fixative that does not
contain an aldehyde. Carnoy, Clarke and methacarn are examples,
but are used only for subsequent paraffin sectioning. (Could one
rehydrate and cut frozen? I can't think why anyone would ever
Glutaraldehyde exists as low polymers. When it reacts with and
cross-links protein molecules, lots of free aldehyde groups
remain. This is probably true also of glyoxal. These tissue-bound
free aldehyde groups will combine covalently with any amino group
offered to them, including terminal and side-chain (lysine) amino
groups of proteins being used as histochemical reagents - that
means all antibodies, all lectins and all enzymes. Your valuable
and highly specific monoclonal primary antibody may bind at sites
that contain basic proteins but not the antigen you're after.
The answer to the aldehyde problem is aldehyde blocking. This is
done by reducing the -CHO groups to -OH with sodium borohydride
or by feeding them bland amino groups (glycine, bovine albumin,
This problem isn't confined to bifunctional aldehyde fixatives.
Antibodies (and lectins) are proteins, and protein molecules
like to hug and kiss one another. Even after a non-aldehyde
fixation, this nonspecific protein-protein adhesion can be
overcome by including irrelevant proteins in the solutions of
dissolved immunoreagents. The classical bland additive is a
non-immune serum from the species in which the secondary antibody
was raised. Skimmed milk and glycine are cheap substitutes.
Some of the anti-autofluorescence methods that Tamara wrote
about are very general and very useful, but (I suspect) rarely
used. They come in two categories: dyes and metals. Either
will provide complete suppression of previously present
fluorescence if used intelligently. Heavy metals drown out
the ability of nearby organic molecules to exercise their
electrons in a way that generates light with a higher
wavelength. Dilute osmium tetroxide (0.2% for 5 min, followed
by 12 hrs or so in running water) is an extremely potent
blocker of autofluorescence, better in my experience than
ferric chloride or mercuric chloride.
Dyes may work by absorbing the exciting light or the
emitted light. (I suspect there's a lot of chemistry here
that I don't know about and could never understand.) The
best ones for squashing autofluorescence seem to be blue
(but don't use aniline blue - it contains a fluorescent
impurity). Blue nuclear stains (haemalum, toluidine blue)
have an obvious anti-fluorescence effect. This is easily
seen by looking with fluorescence microscopy at a slide
stained with H & E or with Giemsa or a similar blood
stain. (Eosin fluoresces quite strongly.)
The word "autofluorescence" is often used rather vaguely, to
mean native fluorescence (e.g. porphyrins, lipofuscin) and
fixative-induced fluorescence. It should not be extended to
include fluorescence due to non-specific binding of reagents
such as primary or secondary antibodies. Nonspecific binding
of this kind has many causes (including fixation, expecially
by glutaraldehyde) and is generally treated by applying a
protein or an amine (glycine, lysine) as mentioned above.
This doesn't provide a direct answer to the original question,
but if you can decide on the cause of the unwanted fluorescence
you can usually come up with a way to reduce it greatly.
The original question.
> >I am trying to colocalize two antigens using two different fluorochromes
> >on Rat frozen brain sections. There is no problem with
> >immunohistochemistry. Except that there are little particles on the
> >tissue that autofluoress under both wavelengths. The autoflorescence is
> >there regardless of the fixative and the antibodies used (I have done all
> >the tests). Is there a way of quenching or blocking this autofluorescence
> >before doing the immuno?
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