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Thank you very much !



Confocal Microscopy List <[log in to unmask]> wrote on 
04/05/2011 06:35:56 PM:

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> 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:
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> >
> > 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
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