Autofluoresence problem

Tom Frey tomfrey at sprintmail.com
Sat May 24 00:36:18 EST 1997


Guy Hermans wrote:
> 
> In article
> <Fergus.Doherty-ya02408000R2205971605060001 at news.nottingham.ac.uk>,
> Fergus.Doherty at nottingham.ac.uk (Fergus Doherty) wrote:
> 
> > Anyone know how to reduce/abolish autofluorescence on formain fixed,
> > paraffin embedded sections of human endometrium?  (Obviously dewaxed before
> > use, but which give a high fluorescent background never having seen any
> > antibody).
> 
> > Fergus.Doherty at nottingham.ac.uk
> 
> In flow cytometry we try to avoid formalin fixation as much as possible -
> it's knows to cause a drastic increase in autofluorescence, creating a
> very high background. Try another fixative!
> 
> Guy
> 
> --
> Guy Hermans, PhD student
>   Ms research Unit              Immunology research group
>   Dr. L. Willems-Institute      Dept. of Physiology, LUC
>   University Campus             University Campus
>   B-3590 Diepenbeek             B-3590 Diepenbeek
>   Belgium                       Belgium
> Voice  ++32(0)11/26.92.07
> Fax    ++32(0)11/26.92.09

Actually, in flow we frequently use paraformaldehyde (dilute formalin)
but studiously avoid glutaraldehyde.  (EM grade glutaraldehyde is
apparently OK and some people use it)  There are some tricks with
reducing agents (sodium borohydride) that have been published for flow
to reduce flourescece, but I wouldn't really bet on that being a
solution for your problem.  Do you know what the autofluorescence of
unfixed material is like?  Also do you know if the deparafinizing
solvent (xylene?) is well removed?  If you want some uneducated
guesswork feel free to communicate directly.



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