Dear fellow Histonetters,

I recently started reusing immunofluorescence after years of
immunoperoxidase detection systems. I have encountered a problem with
"background" fluorescence on both paraffin and polyethylene glycol
sections. Under fluorescence illumination, these sections appear to
"fluoresce" all over the sections, even if I have not applied any
fluorochrome. This effect is more marked on the fluorescein channel, than
the rhodamine or the UV channel. I can see my immunostaining after
immunolabelling, but it has to stand out against a general red or green
background on the sections, which probably obscures faint signals.

I have checked sections of tissues fixed with different fixatives (aldehyde
and non-aldehyde), and its not linked to any particular fixative. I don't
observe this effect when doing immunofluorescence on both cell cultures or
fresh frozen tissue section fixed by methanol or formaldehyde. My sections
were mounted in PBS/glycerol with different antifading agents (no
correlation there) or unstained sections were also dehydrated and mounted
in Eukitt.

I am not sure whether this is really fluorescence or some kind of "light
scattering" on paraffin and PEG sections. So what is happening here ?? Has
anyone encountered similar problems and is there a way to reduce this
effect ??

Thanks

Paul
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Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel. 03.88.35.85.04  Fax. 03.88.24.04.61
========================klosen@neurochem.u-strasbg.fr=========================