Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Endogenous biotin in mast cells?
Do mast cells contain any endogenous biotin? They are often falsely positive in immunostaining methods that use avidin.
Mast cells bind avidin nonspecifically because of ionic attraction between avidin (a basic protein) and heparin (acid polysaccharide in MC granules). This results in false positive staining by ABC. The cure is to use the ABC reagent at pH 9.4. For more information see Bussolati, G & Gugliotta, P 1983. Nonspecific staining of mast cells by avidin-biotin-peroxidase complexes (ABC). J. Histochem. Cytochem. 31: 1419-1421.
John A. Kiernan, Department of Anatomy & Cell Biology,
The University of Western Ontario,
LONDON, Canada N6A 5C1
Bussolati and Gugliotta (J. Histochem. Cytochem., 31(12): 1419-1421, 1983) described binding of ABC to mast cells. They believed this to be due to both the binding of avidin basic residues as well as peroxidase to the sulphate groups of heparin. They showed that binding could be prevented by using the ABC solution at a pH of 9.4. This high pH does not affect either previous binding or localisation of antibody or the affinity of biotin for avidin.
They also showed that the nonspecific binding of avidin could be blocked by a 30 minute pretreatment of sections with a synthetic basic polypeptide such as poly-L-lysine (0.01% in PBS, pH 7.6).
Tony Henwood, Senior Scientist
Royal Prince Alfred Hospital