Quantitative Studies of Immunofluorescent Staining

Abstract

Unwanted staining reactions in tissue immunofluorescence (IF) entail two mechanisms, notably the nonimmunologically mediated nonspecific staining (NSS) and undesired specific staining reactions (USS) of unwanted antibodies. Indirect IF titrations and assays for fluorescein and anti-Ig antibody protein employed might afford procedures for evaluating the efficacy of absorption with the tissue sediments. Absorption of anti-immunoglobulin conjugates with tissue sediments removes the USS. Formalin fixation reduced reactivity of tissue antigens but had no detectable effect on the reaction of the heterophilΕ antibodies. The addition of bovine serum albumin to the buffered saline diluent stabilizes conjugates and sera but has no significant effect on specific or nonspecific staining at concentrations up to 4%. It is recommended on the basis of these studies that FITC conjugates used in indirect IF studies of tissue antibodies be absorbed with tissue sediments and diluted in 4% bovine serum albumin.