reducing autoflourescence in paraffin embedded adipose tissue?

[Richard Green-2]

Any recommendations on reducing autoflourescence in paraffin embedded  
adipose tissue? We are looking into TSA to amplify. Any suggestions  
folks have is muchly appreciated. Thanks
-Richard green

[Martin Wessendorf-2]

Richard Green wrote:
> Any recommendations on reducing autoflourescence in paraffin embedded
> adipose tissue? We are looking into TSA to amplify. Any suggestions
> folks have is muchly appreciated. Thanks

Dear Richard--

What color is the autofluorescence and through what filters is it
visible?  If it's punctate and visible through FITC, rhodamine and CY5
filters but is yellow under wide-band UV filters, it's lipofuscin.  That
can be reduced by incubation with CuSO4.

It might also be aldehyde-induced fluorescence if its not punctate but
also with broad ex-em spectra.  That can be reduced with NaBH4.  This is
a particular problem with glutaraldehyde fixation.

Send me a note if you have trouble finding references on the methods.

Good luck!

Martin Wessendorf

--
Martin Wessendorf, PhD   [phone] (office)
Associate Professor      [phone] (lab)
Dept Neuroscience      [phone] (FAX)
Univ Minnesota        e-mail: martinw(at)umn.edu

[Laszlo Komuves]

I have tested several published reagents/treatments not to avail.

The best solution is to image using spectral deconvolution (the NUANCE system from CRi has been successfully used in our lab for the past two years).

Moreover if you use TSA which will create a very robust and almost non-fading signal. Therefore carefully controlling the exposure will capture the real signal as the signal-to-noise ratio will be quite high (assuming that you do not have an amplified background do to non-specifc TSA signal).

Best,

Laszlo

László G. Kömüves PhD, Senior Scientist (Technology) | Center for Advanced Light Microscopy (CALM) | Department of Pathology | GENENTECH, Inc. | One DNA Way South San Francisco CA 94080-4990 | Phone: [phone] | E-mail: [hidden email]

On Oct 28, 2009, at 5:22 PM, Richard Green wrote:

Any recommendations on reducing autoflourescence in paraffin embedded adipose tissue? We are looking into TSA to amplify. Any suggestions folks have is muchly appreciated. Thanks
-Richard green