Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Glutaraldehyde and immunohistochemistry
Does glutaraldehyde fixative (4% paraformaldehyde, 0.5% glutaraldehyde) interfere with fluorescent immunohistochemistry?
Glutaraldehyde, because of its reactivity and speed, can seriously interfere with antibody binding and lectin binding causing considerable non-specific binding. It is also difficult to remove excess glutaraldehyde from tissue components. I would not recommend it's use for such studies, as in my hands the results have been inconsistent.
Tissues fixed in glutaraldehyde exhibit increased autofluorescence, which is probably due to glutaraldehyde-amino acid compounds that are formed as part of the fixative action. Glutaraldehyde also introduces free aldehyde groups into the tissue, and these will bind any protein reagents that are applied. The nonspecific binding of antibodies can be reduced by pretreatment with a
blocking protein (such as bovine albumin, or serum from the species in which a secondary antibody was raised). Before the blocking treatment it is advisable to do a chemical aldehyde blockade (Histochemistry textbooks contain several methods).
John A. Kiernan