background autofluoresence in heart frozen sections

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R Bishop
R Bishop's picture
background autofluoresence in heart frozen sections

Im trying to do immunofluoresence in frozen mouse heart sections with a secondary cojugated to Alexa 488.  The problem is the backgound in the green channel is enormous in the secondary only slides.  I tried it again and it appeared even when I added primary antibody only (no secondary-labeled antibody). As anyone else experienced this problem? Oh and the same did not occur in muscle from the same mice?
 
Rus

Arvind Singh Pundir
Arvind Singh Pundir's picture
it may be due to the

it may be due to the autofluorescence i.e, natural fluorescence or fixative induced fluorescence, in the link below there are methods of reducing the autofluorescence and details about different autofuorescence agents
Autofluorescence : Causes and Cures
www.uhnresearch.ca/facilities/wcif/PDF/Autofluorescence.pdf

The FFM
The FFM's picture
I think there is much more

I think there is much more lipofuscin in heart tissue than in muscle and this will crank up your autofluorescence.
Do you have access to a speactral confocal or fluroscence microscope which will allow you to lineraly unmix the background autofluorescence from the Alexa488 signal?
here are some papers that deal with lipofuscin autofluoreascence
www.jhc.org/cgi/content/abstract/43/7/727  or  the pdf www.jhc.org/cgi/reprint/43/7/727.pdf
www.jhc.org/cgi/content/full/47/6/719
www.iovs.org/cgi/content/abstract/43/7/2435