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
HTL(ASCP)HT,MT
Bozeman MT
----- Original Message -----
From: <anh2...@med.cornell.edu>
To: "Bob Nienhuis" <bob.nienh...@gmail.com>; "Gayle Callis"
<gayle.cal...@bresnan.net>
Cc: <histo...@pathology.swmed.edu>
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.nienh...@gmail.com>
Date: Fri, 12 Dec 2008 17:16:27
To: Gayle Callis<gayle.cal...@bresnan.net>
Cc: <histo...@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.cal...@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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