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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello, 

The issue has been discussed before and you may check the archive. The main
thing is to identify the source of auto-fluorescence.

Here are few options: 
1) Chemical treatment using CuSO4 or Sudan black (Schnell SA, Staines WA,
Wessendorf MW.; J Histochem Cytochem. 1999. 
Check also: Baschong et. al., J. Histochem. Cytochem. 2001 49(12):1565-1571.

2) Use spectral imaging if you have access to one such instrument. If you
can get a decent spectra of your signal, spectral imaging perform well in
removal of autofluorescence.

3) You may be able to use another laser line to image the auto-fluorescence
signal and perform some image math. to subtract autofluroscence from your
signal channel. You need a control slide. But it is very effective and can
be done online with most confocals.

4) I read sometime ago using a light-box at low temperature to bleach out
autofluorescene prior to staining. But I do not remember the references
(sorry).

Good luck!

Xuejun

Xue-jun Sun, Ph.D.
Dept. Exp. Oncology
Cross Cancer Institute
11560 University Ave,
Edmonton Alberta T6G 1Z2
Canada
Phone: (780) 432-8898
Fax:     (780) 432-8425

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Soumitra Ghoshroy
Sent: Friday, October 06, 2006 8:12 AM
To: [log in to unmask]
Subject: brain autofluorescence

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Everyone,

We have an user who wants to label an enzyme in hypothalamus using
immunoflurescence. We used Alexa Fluor 647 as secondary in paraffin embedded
tissue and discovered that the tissue is autofluorescing in almost every
laser
line. I am not at all familiar with brain tissue.  We also used unlabeled
deparaffinized sections and looked at it and the autofluorescence level is
extremely high. So I would like to get some advice on how to image it. Is
there
anything we are not doing right? The tissue was fixed with 4%
paraformaldehyde.

Thanks in advance.

Soumitra


****************************************************************************
***
Soumitra Ghoshroy 
College Associate Professor, Biology
Director, Electron Microscopy Lab
New Mexico State University
Las Cruces, NM 88003
Tel: 505-646-3268 (office)
     505-646-3283 (lab)
Fax: 505-646-3282
http://biology-web.nmsu.edu/ghoshroy/ghoshroy.htm 
http://emldata.nmsu.edu


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