RE: Quenching autofluorescence

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From:Tamara Howard <>, Histology listserver <>
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Lilith - 

	There are quite a few things you can try. Someone has already
suggested Cu sulfate and Sudan Black...I've had some success with treating
the samples with a dilute (~0.5% aq.) Toluidine Blue stain (your local EM
lab should have some stock on hand). I've also seen protocols for using
Pontamine Sky Blue and some other dyes the same way...I just don't have
the stains on hand so have never tried them. You do the "stain" before the
immuno - it shouldn't mess up your antigens.

	Depending on what is causing the autofluorescence, you may be able
to kill or reduce it with a glycine (0.3M) wash or with a light Na
borohydride treatment. There is another...with one of the ammonium salts,
I think...but I'm drawing a blank. If you are interested I can organize an
archeological expedition to my files and see if I can dig it up - although
I'll bet that Dr. Keirnan knows it right off :)

Tamara Howard

>Dear Colleagues,
>I am trying to colocalize two antigens using two different fluorochromes
>on Rat frozen brain sections. There is no problem with 
>immunohistochemistry. Except that there are little particles on the
>tissue that autofluoress under both wavelengths. The autoflorescence is
>there regardless of the fixative and the antibodies used (I have done all
>the tests). Is there a way of quenching or blocking this autofluorescence
>before doing the immuno?
>Thanking you in advance for your help,

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