Re: [Histonet] immunofluorescence on paraffin sections (looking for a detailed protocol)


In my hands, immunofluorescence on paraffin is a very frustrating and 
unrewarding task. I am sorry to have to say this.  Fixatives that 
crosslink tissue (para- and formaldehyde) cause massive collagen 
crosslinking.  Collagen is  inherently highly auto-fluorescent.  If the 
bundles of collagen are cross-linked the autofluorescence is increased 
to a point in which it strongly interferes with most fluorochromes used 
for immunofluorescence.
There may be some ways to reduce the autofluorescence of cross-linked 
tissue and I am sure you would be receiving the best advice in this 
forum.  However, I thought it would be important to mention the major 
setbacks of the technique just incase you may have not been aware of 

When possible, use frozen section of fresh unfixed tissue or even 
better, whole explants if and when this possibility exists (tissue 
slices, retinas,peritoneum, etc)

Dr.Rocio Sierra-Honigmann
Engineered Wound Repair Laboratory
Cedars Sinai Research Institute
Davis 1091

On Jan 27, 2005, at 4:00 AM, Marco Prunotto wrote:

> I hope someone can send me a good protocol to make immunofluorescence 
> on paraffin sections.
> I saw that there is a  an heat induced antigen retrieval method with 
> Tris 100 mM and 5% urea..
> Can someone send me a detail protocol?
> thanks a lot
> best regards
> -------------------------------------
> Marco Prunotto, PhD
> Pathology Dept., University of Geneva - CH
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