Mixed glycosidase pretreatment reduces nonspecific binding of antibodies to frozen tissue sections.
Indirect immunohistochemical studies of frozen mouse tissues with mouse monoclonal antibodies yield, in general, suboptimal results primarily because of indiscriminate binding of secondary antibody to all mouse immunoglobulins, i.e., to the monoclonal reagent and to endogenous immunoglobulin nonspecifically trapped in the tissue. To reduce this nonspecific staining, frozen sections of mouse kidney were treated enzymatically. Optimal results were obtained following a 2 hr treatment with 20 mg/ml of mixed glycosidases (MG). This treatment reduced the nonspecific background staining of the interstitial spaces and blood vessels, but did not affect the reactivity of structurally bound immunoglobulin G (IgG) in the glomeruli or alter the reactivity of mouse renal tissue to the monoclonal antibody that recognizes an oligosaccharide antigenic determinant (SSEA-1). Eluates from enzyme-treated frozen tissue sections contained normally immunoreactive IgG in the form of dimers. These data indicate that MG treatment of frozen sections could be safely used to reduce the content of nonstructurally bound immunoglobulins in frozen tissues and thus improve the visualization of specific monoclonal antibody binding.
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D005615:Freezing; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006651:Histocytochemistry; D007074:Immunoglobulin G / Q000378:metabolism; D007163:Immunosorbent Techniques; D007668:Kidney / Q000166:cytology* / Q000276:immunology; D008722:Methods; D051379:Mice
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D006026:Glycoside Hydrolases