[Histonet] Autofluorescence and literature for getting rid of the problem

[WILLIAM DESALVO]

I would suggest using a sigma-aldrich product, Evans Blue (E2129). This product can be used as a counterstain in immunohistochemistry when using FITC. After staining for immunofluorescence, dip sections in a 0.1% (w/v) in water solution of Evans Blue for 5-10 minutes. Rinse well in fresh PBS or water before coverslipping. Reference: Immunocytochemistry, Theory and Practice, p. 82 (1988). Quick and very easy.


William DeSalvo, B.S., HTL(ASCP)




 
> From: gayle.callis <@t> bresnan.net
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Tue, 19 Oct 2010 14:06:29 -0600
> Subject: [Histonet] Autofluorescence and literature for getting rid of the problem 
> 
> Richard, 
> 
> 
> 
> You will find what you need in a free pdf on autofluorescence from Wright
> Imaging Facility in Toronto Canada. They have a website, then download
> Autofluorescence: Causes and Cures. Also, if you can't get rid of
> autofluorescence , use a near infrared fluorophore e.g. Alexa 750. There is
> no autofluorescence seen in the NIR range. Just Google the title and it
> will come up instantly. They also have a pdf on Mounting Media for
> fluorescence work.
> 
> 
> 
> 
> 
> For the little FFPE fluorescent work we do, you can try 100 to 300 mM
> glycine in TRIS buffer pH 7.4 for 20 to 30 minutes before embarking on
> immunostaining. Glycine binds free aldehydes to reduce the
> autofluorescence but doesn't always work 100%. It may reduce the problem
> but not totally eliminate it. 
> 
> 
> 
> There are other references on getting rid of autofluorescence, one for GFP,
> a review, but it applies to FFPE tissue even not containing GFP. I will be
> happy to send the pdf if you wish. 
> 
> 
> 
> Gayle M. Callis
> 
> HTL/HT/MT(ASCP) 
> 
> Bozeman MT 
> 
> 
> 
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