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| Date: | Mon, 28 Jan 2013 12:40:53 -0600 |
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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: (612) 626-0145
Assoc Prof, Dept Neuroscience lab: (612) 624-2991
University of Minnesota Preferred FAX: (612) 624-8118
6-145 Jackson Hall, 321 Church St. SE Dept Fax: (612) 626-5009
Minneapolis, MN 55455 e-mail: [log in to unmask]
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