Quenching glutaraldehyde autofluorescence

Raymond B. Hester (rhester@jaguar1.usouthal.edu)
Wed, 19 Apr 1995 07:39:45 -0500 (CDT)

A user of our image analyzer is fixing 5 u sections of cardiac tissue
with glutaraldehyde and staining with an anti protein kinase C antibody.
They tried several fixatives and glutaraldehyde seems to work best with
the PKC antigen. When they assay using a biotin avidin HRP technique, it
works well, i.e., good reactivity with antibody and low background in
controls. They have a three-tiered system: the primary ab, a
secondary conjugated with biotin, a third conjugated with avidin horse-
radish peroxidase. When they try substituting a fluorescence-tagged
antibody for the HRP, it doesn't work because the autofluorescence of the
(glutaraldehyde-fixed) tissue is very high. They are going to reassess the
fixation alternatives to see if there is one that will retain reactivity
of the PKC epitopes and yet produce much lower autofluorescence.

In case the other fixatives also destroy the reactivity of PKC with
anti PKC antibody and they have to stick with glutaraldehyde
fixation, does anyone have any experience blocking the
autofluorescence of glutaraldehyde-fixed tissue sections?

Does anyone have experience with fluorescence assays detecting PKC
in fixed tissue and, if so, what do you use to fix the tissue?

Thanks for your help.

Ray

++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Raymond B. Hester, Ph.D.
Flow Cytometry Lab
The University of South Alabama "Life is too short to be small."
Mobile, AL 36688-0002
Email: rhester@jaguar1.usouthal.edu Disraeli
Voice: (334) 460-6029
FAX: (334) 460-6073
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