Re: Background fluorescence

<< Previous Message | Next Message >>
Date:Tue, 5 Oct 1999 12:27:56 +0100
Content-Type:text/plain; charset=us-ascii

Hello Paul,

You are not alone!  Your enquiry immediately caught my attention, as I have just
recently had a similar experience with formaldehyde fixed paraffin sections.
The strong autofluorescence I was detecting was coming mainly from fixed red
blood cells in the tissue sections which was as strong as or stronger than the
PCNA label I was trying to detect.  I am working with a laser scanning cytometer
hoping to be able to quantify a number of different proliferation and apoptosis
markers in drug treated tumor models in mice.
My background is in flow cytometry from which I know that fixation with
formadehyde does increase autofluorescence in single cells especially in the
channel normally used to measure FITC (green fluorescence).  My first
explanation for what I saw in the tissue sections was that this is what is
happening here.  Added to this is the fact that I was working with 5 micron
sections, (though even in 3 micron human tonsil sections the red blood cells
were also highly autofluorescent.)  which probably helped augment the background
fluorescence problem.

I would be very interested to know if other Histonetters have words of wisdom/
possible solutions to this problem.

Kathie Woods-Cook
Novartis Pharma AG
CH4002 Basel, Switzerland

Paul Klosen <> on 05.10.99 12:01:37

cc:    (bcc: Kathie A Woods-Cook/PH/Novartis)
Subject:  Background fluorescence

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 ??


                                   (o -) O
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.  Fax.

<< Previous Message | Next Message >>