Re: [Histonet] Heat and autofluorescence

From:Merced Leiker

I would say no, since it's routine to dry them at 60 degrees 1 hour.  What 
tissue was this?  What was it fixed in? We've had similar autofluorescence 
issues doing exactly the same thing your researcher is doing with EGFP.  We 
were looking at skeletal muscle, however, which is highly autofluorescent 
in just about every visible light channel.  EGFP-containing cells/tissues 
need to be fixed in 4% PFA.  Avoid 70% ethanol as it will allow this 
protein to leak out.  We had a hard time in general with EGFP.


--On Thursday, October 09, 2008 8:16 PM +0000 wrote:

> I have a researcher in my lab that transfected cells with EGFP, injected
> them into mouse and then 3 weeks later harvested the tissue and frozen it
> in OCT as usual.  After sectioning, he air dried the slides for 1 hour
> and then dried them at 60 degrees C for 1 hour.  He was hoping just to
> see his EGFP infected cells which are bright green, however, the
> autofluorescence is everywhere - both with GFP and Rhodamine filters.
> Our question is - did the heating in the oven cause increased
> autofluorescence?
> Cathy
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Merced M Leiker
Research Technician II
354 BRB (Lee Lab) / 140 Farber Hall (mail)
School of Medicine and Biomedical Sciences
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214
Ph: (716) 829-6033
Fx: (716) 829-2725

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you must first take off yours.

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