Re: autofluorescence (and how to store OsO4)

From:"J. A. Kiernan" <>

On Thu, 5 Jul 2001, Rudolf Schicho wrote:

> Does somebody have a good idea how to get rid of autofluorescence in the
> jejunum (rat; fixed in 4% paraformaldehyde and 1.2% picric acid; cryo
> sections)? It seems to be packed with autofluorescent cells. 

Are these enterochromaffin cells? They contain lots of
5HT and it gives a fluorescent reaction product with
formaldehyde (which is the only active ingredient of
your fixative if it is adjusted or buffered to a near
neutral pH).

The best way to kill fluorescence in sections is to
immerse in a dilute solution (e.g. 0.1% in water) of 
osmium tetroxide for 5 to 10 minutes. This must be
followed by a Really Thorough Wash, such as running
(well, trickling) tap water ideally overnight. Half an
hour isn't enough. 2 hours is OK for thin (5 um)

The next bit is rather long and only indirectly 
related to your enquiry about autofluorescence.

An expensive OsO4 solution can be re-used until it 
goes dark (reduced to OsO2 by bits of dirt etc).
There's a lot of superstition about storing osmium
tetroxide solutions. They are _not_ affected by light,
and they decompose only when contaminated with 
organic reducing agents (filter to remove bits of 
sections; don't let any alcohol get in it). The
only reason to put an OsO4 solution in the fridge
is to slow down reduction by contaminants. (These
statements are supported by books on osmium chemistry
as well as by my casual observations.)

The type of bottle and its bung are the most important 
factors in preserving your osmium tetroxide. A ground 
glass stopper is the worst, because OsO4 vapour easily
finds its way out and blackens nearby plastic and
other surfaces. (You can't grease the ground glass
because OsO4 is about 200X more soluble in hydrocarbons
than in water.)  A cork is a similarly ineffective way
to keep OsO4 in its bottle. The most effective closure
is a Black Rubber Stopper (or Bung), which fits into the 
neck of a glass bottle tightly enough to prevent loss of
OsO4 vapour. I learned this from M. Gabe's "Histological
Techniques" (Paris: Masson, 1976). Other colours of
rubber are also OK, but they all react with OsO4 vapour
and blacken. You don't see this with black rubber!
Polyethylene stoppers and bottle-cap liners are almost
useless for restraining the escape of OsO4.

'nuff said for the time being.

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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