Re: ECM Autofluorescence blues - any solutions?

From:Kathleen Spencer


I have possibly solved my autofluorescing problem. I am working on it 
now, my slides are soaking in a solution of 1mM CuSo4 (copper sulfate) 
in a 50mm ammonium acetate buffer, pH 5.0, as I type this. BUT, what 
auto fluoresces in my tissue (we now think) is lipofuscin in aged rat 
brain, so I don't know if this will help you at all. They also 
suggested  using Sudan Black B in 70% ethanol.
Just wanted to let you know, since I read your new post.
ref. is The Journal of Histochemistry and Cytochemistry 1999 volume 
46(6): 719-730
Hope you get some answers to your specific problem soon. I know how you 
feel. I have been so frustrated with this. We are 99% sure that that was 
our problem. We'll see!


on Tuesday, October 15, 2002, at 09:45 AM, Gail Donegan wrote:

> Hi everybody,
> Thanks to everyone for the help and suggestions regarding what I 
> mistakenly assumed was non-specific binding of my secondary antibody. 
> Now I find I have a different problem altogether...
> The extracellular matrix (ECM) laid down by my human smooth muscle 
> cells fluoresces red at 594nm (so fluorescently staining with an 
> antibody that fluoresces red is pointless) and which also fluoresces 
> green at 495nm (so using a green fluorescing antibody is no good 
> either).
> I've tried using 100mM - 500mM Glycine, which worked for Andrea who 
> kindly responded to my plea last time I was in touch.
> Does this mean that fluorescently staining adsorbed ECM is not possible 
> or is someone out there doing it?
> Any suggestions gratefully received,
> Thanks,
> Gail
> Gail Donegan
> Department of Clinical Engineering,
> The University of Liverpool,
> Duncan Building,
> Daulby Street,
> Liverpool,
> L69 3GA.
> U.K.
> Tel:    +44(0)151 706 5198
> Fax:    +44(0)151 706 5803

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