[Histonet] Re: Reduction of autofluorescence using glycine

[anh2006 <@t> med.cornell.edu]

No, because the glycine acts by reducing the autofluorescene of the free aldehydes (maybe Dr. Kiernan or another knowledgeable person in the chemistry can tell us precisely how) rather than reducing the binding of other staining components to the aldehydes.

-----Original Message-----
From: Bob Nienhuis <bob.nienhuis <@t> gmail.com>

Date: Fri, 12 Dec 2008 17:16:27 
To: Gayle Callis<gayle.callis <@t> bresnan.net>
Cc: <histonet <@t> pathology.swmed.edu>
Subject: Re: [Histonet] Re: Reduction of autofluorescence using glycine


If this works by binding free aldehyde groups that attach to antibodies/ or
fluorochromes,  or biotinylated whatever. shouldn't it also work for DAB or
ABC immunolabeling and
reduce background labeling?

Bob
UCLA / VA Medical Center

On Fri, Dec 12, 2008 at 2:08 PM, Gayle Callis <gayle.callis <@t> bresnan.net>wrote:

> To reduce aldehyde induced autofluorescence, you can use 100 - 300 mM
> glycine in pH 7.4 buffer.  TRIS buffer or even Dulbeccos PBS will work.  You
> rehydrate the section and then immerse into the glycine solution for 20
> minutes, maybe even longer.  Glycine works by getting rid (binding?) of free
> aldehyde groups.  You can either treat the tissue prior to processing (after
> fixation) by immersing for an hour or so, but we simply did the glycine
> treatment on individual sections.  It worked best for us when we did a short
> length fixation in NBF.
>
> This has been discussed at length on Histonet in the past, so do an archive
> search.  One person put a summary together on various methods and what
> worked best for him.
>
> There are other methods for getting rid of autofluorescence although some
> are less successful than others and one is made from a chemical that is
> explosive.   Try IHCworld website, fluorescence topics  or Google access
> this discussion written by Wright Cell Imaging Faculty, Toronto Western
> Research Institute, titled:  Autofluorescence, Causes and Cures, a must read
> on the subject.
>
> Another trick is to use fluorophores in the near infrared range, the camera
> sees the fluorescence but no autofluorescence and you cannot see this red
> fluorophore with the naked eye.  Alexa 750 will work if you have the filters
> and excitation wavelength available.
>
> Good luck
>
> Gayle M. Callis
> HTL(ASCP)HT,MT
> Bozeman MT
>
>
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