*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear JP--

 From your description, it might be lipofuscin.  In my limited 
experience, mouse tissue has a lot of it.  If that's what it is, it will 
appear yellow/orange with a wide-band UV filter, green through a 
fluorescein filter, red through a rhodamine filter and will also be 
visible through a Cy5 filter.  It will be very resistant to 
photobleaching.  It will remain fluorescent after treatment with NaBH4 
or oxidizing agents or after lipid extraction.

You can deal with lipofuscin one of at least 3 ways:

1)  Don't use fluorescence--use immunoperoxidase methods.

2)  Treat with Cu++.  Cu++ ion will quench much of the autofluorescence 
of lipofuscin, but it will also somewhat reduce the fluorescence of the 
Cy3.

3)  Treat with Sudan Black.  Sudan Black binds lipophilic compartments 
and, being black, quenches lipofuscin.  It's not compatible with 
xylene-based mounting media, though.

Steve Schnell, Bill Staines and I have a paper on this:
Schnell SA, Staines WA, Wessendorf MW.  Reduction of lipofuscin-like 
autofluorescence in fluorescently labeled tissue.  J Histochem Cytochem. 
1999 47(6):719-30.

Good luck!

Martin Wessendorf

  On 4/5/2011 8:06 PM, Jean-Pierre CLAMME wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> I'm looking for someone having experience with confocal imaging of liver
> tissue. I'm imaging mouse liver tissue and I have some issues with
> autofluorescence in the cy3 channel coming from structure appearing like
> vesicle in hepatocytes. Those vesicles mainly show up in the Cy3 channel
> but are also visible in the FITC channel (depending on the power I use).
> A spectral image with excitation at 488, shows a broad signal with a
> maximum around 580 nm.
> Could someone comment on the origin of this fluorescence ? Is it possible
> that it is lipofuscine ?
>
> Thank you,
>
> JP



-- 
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
University of Minnesota             Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: (612) 626-5009
Minneapolis, MN  55455                    e-mail: [log in to unmask]