Glutaraldehyde and sodium borohydride reduction

[kspencer007]

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Hello;
        Please indulge a cell biologist trying to understand chemistry...
        We are discussing the differences between glutaraldehyde and formaldehyde fixation for immunofluorescence. When fixing with glutaraldehyde (0.1% glut with 3% PFA), we follow the fixation with sodium borohydride reduction. I understand that this converts the unused aldehyde groups to unreactive hydroxyls. Why is this step not required for formaldehyde fixation? Merely because PFA has one aldehyde group and Glut has two on the ends exposed after polymerization?
        Thanks.
        Kathy


Kathryn R. Spencer, PhD
The Scripps Research Institute
10550 N. Torrey Pines Road, DNC 210
La Jolla, CA 92037

[ae4710]

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Here is a very nice summary of the ins and outs of aldehyde fixation:

http://publish.uwo.ca/~jkiernan/formglut.htm 

Sincerely,
Will

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Kathryn Spencer
Sent: Monday, January 28, 2013 1:11 PM
To: [hidden email]
Subject: Glutaraldehyde and sodium borohydride reduction

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Hello;
        Please indulge a cell biologist trying to understand chemistry...
        We are discussing the differences between glutaraldehyde and
formaldehyde fixation for immunofluorescence. When fixing with
glutaraldehyde (0.1% glut with 3% PFA), we follow the fixation with sodium
borohydride reduction. I understand that this converts the unused aldehyde
groups to unreactive hydroxyls. Why is this step not required for
formaldehyde fixation? Merely because PFA has one aldehyde group and Glut
has two on the ends exposed after polymerization?
        Thanks.
        Kathy


Kathryn R. Spencer, PhD
The Scripps Research Institute
10550 N. Torrey Pines Road, DNC 210
La Jolla, CA 92037

[Martin Wessendorf-2]

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Dear Dr. Spencer--

On 1/28/2013 12:10 PM, Kathryn Spencer wrote:

> Please indulge a cell biologist trying to understand chemistry...
> We are discussing the differences between glutaraldehyde and formaldehyde fixation for immunofluorescence. When fixing with glutaraldehyde (0.1% glut with 3% PFA), we follow the fixation with sodium borohydride reduction. I understand that this converts the unused aldehyde groups to unreactive hydroxyls. Why is this step not required for formaldehyde fixation? Merely because PFA has one aldehyde group and Glut has two on the ends exposed after polymerization?
> Thanks.
> Kathy

Treatment with NaBH4 reduces autofluorescence (--the autofluorescence
apparently results from Schiff bases formed by reaction of aldehydes
with primary amines).  Glutaraldehyde induces autofluorescence much more
strongly than formaldehyde, although in my experience, treating with
NaBH4 measurably reduces background for formaldehyde-fixed tissue as well.

Good luck!

Martin
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Michael Schell-2]

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Reducing with borohydride after glut fixation reduces the double-bonds that form between glutaraldehyde and amine groups in the tissue.  As Martin just posted, this treatment reduces tissue autofluorescence  (but often does not eliminate it completely) .

In addition, reducing those bonds changes antigenticity--usually for the better.  Many immunogens are injected in their reduced form, so you aim to recreate that shape i the tissue by reducing it too.  Some "background" antigenicity may be due to double bond-containing epitopes, so you want to eliminate those.  Thirdly, permeability/penetration of the tissue is somewhat improved because the reduced bonds can rotate.

Michael

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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>
> Hello;
> Please indulge a cell biologist trying to understand chemistry...
> We are discussing the differences between glutaraldehyde and formaldehyde fixation for immunofluorescence. When fixing with glutaraldehyde (0.1% glut with 3% PFA), we follow the fixation with sodium borohydride reduction. I understand that this converts the unused aldehyde groups to unreactive hydroxyls. Why is this step not required for formaldehyde fixation? Merely because PFA has one aldehyde group and Glut has two on the ends exposed after polymerization?
> Thanks.
> Kathy
>
>
> Kathryn R. Spencer, PhD
> The Scripps Research Institute
> 10550 N. Torrey Pines Road, DNC 210
> La Jolla, CA 92037