More on Re: [Histonet] Reduction of autofluorescence using glycine

Gayle Callis gayle.callis <@t>
Sat Dec 13 17:45:47 CST 2008

For information on how (chemically) aldehyde fixatives cause 
autofluorescence and also a link to JAK  e.g. John A Kiernan in Histonet 
archives, access the Wright Cell Imaging Facility, Toronto Western Resedarch 
Institute.  This website is linked via www.IHCworld and simply found by 
Googling aldehyde induced autofluorescence.  You will find protocols for 
reducing fixative induced fluorescence, and a tutorial on natural to 
aldehyde induced autofluorescence, plus a great deal more on the subject. 
There is also an excellent reference list.

As for a quick chemical explanatioon on how the aldehyde groups are gotten 
rid of is "by reducing the -CHO groups to -OH with sodium borohydride or as 
they stated, "feeding [the aldehyde groups] bland amino groups" e.g. glycine 
or lysine.

This has been discussed at length on Histonet in the past, and by searching 
with key words, you will probably pick up John Kiernan's comments, plus a 
reference on a Review of Autofluorescence.   If anyone needs the latter 
publication, I will be happy to send via personal email.

As for increased background with ABC methods or DAB, Jules Elias's book says 
antibodies can bind to free aldehyde groups, and the tissue can be 
pretreated in the same way as labs do when working with immunofluorescence. 
One would need to determine if the background is caused by another source 
(endogenous biotin, nonspecific binding of antibodies to tissue 
immunoglobulins, or overdevelopment of DAB) so if the pretreatment to get 
rid of free aldhehydes doesn't work and background still exists - then the 
sleuthing continues.   Gluteraldehyde is the worst offender, along with long 
fixation in NBF, or warm temperatures during NBF fixation.

Gayle M. Callis
Bozeman MT

----- Original Message ----- 
From: <anh2006 <@t>>
To: "Bob Nienhuis" <bob.nienhuis <@t>>; "Gayle Callis" 
<gayle.callis <@t>>
Cc: <histonet <@t>>
Sent: Friday, December 12, 2008 7:19 PM
Subject: Re: [Histonet] Re: Reduction of autofluorescence using glycine

> 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>>
> Date: Fri, 12 Dec 2008 17:16:27
> To: Gayle Callis<gayle.callis <@t>>
> Cc: <histonet <@t>>
> 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>>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
>> Bozeman MT
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