In article <Pine.GSO.3.94.980123103211.10068A-100000 at mars.its.yale.edu> Tom Fischer <tmf3 at pantheon.yale.edu> writes:
>Instead of using formaldehyde, try *fresh* paraformaldehyde. The
>formaldehyde off the shelf tends to autofluoresce, as does old (one day or
>more) paraformaldehyde.
The above statement is is not true.
>> Does anybody know a method how to reduce the fluorescent
>> background?
The easiest trick, in my experience, is to use a fluorescent
label that is excited by green light and emits orange-red.
Rhodamines and Texas red come in this category. If you must
use fluorescein, the autofluorescence is less troublesome if
you excite with near UV rather than blue, with a colourless
barrier filter. The autofluorescence will then be blue, and
fluorescein yellow - a bigger spectral difference than when
the usual filter set for fluorescein is used.
You can kill all fluorescence in a section by putting the
hydrated slide in 0.5% osmium tetroxide for 5 minutes, then
washing for several hours in running tap water. (I can send
references if you need them.) It is important to remove all
traces of osmium before doing the fluorescent staining. Of
course, there is no guarantee that the OsO4 will not oxidize
something in the epitope that you are trying to detect, but
that is easy to check with a known positive control.
John A. Kiernan
Department of Anatomy
Univ. of Western Ontario
LONDON, Canada N6A 5C1
