Postfixation detergent treatment for immunofluorescence suppresses localization of some integral membrane proteins

Abstract

Immunofluorescence microscopy of cultured animal cells is often performed after detergent permeabilization of formaldehyde-fixed cellular membranes so that antibodies may have access to intracellular antigens. A comparison was made of the ability of several detergents, after formaldehyde fixation, to affect localization of intracellular proteins or to permeabilize different organelles to antibodies. Saponin, a detergent-like molecule that can permeabilize cholesterol-containing membranes, was also used. Four monoclonal antibodies were found to have a bright, discrete fluorescence localization with saponin alone, but were almost undetectable when the cells were treated with nonionic detergents such as Triton X-100 or NP-40. These immunoglobulin G antibodies included two against lysosomal membrane glycoproteins, one against an integral membrane protein found in the plasma membrane and endocytic vesicles, and one against a membrane protein in the endoplasmic reticulum and the nuclear envelope. However, antigens localized in mitochondria and the nucleus required the use of a detergent such as Triton X-100 for their detection. The detection of a number of other membrane or cytoplasmic proteins was unaffected by Triton X-100 treatment. It was concluded that nonionic detergents such as Triton X-100 cause artifactual loss of detection of some membrane proteins, and saponin is a favorable alternative reagent for immunofluorescence detection of intracellular membrane antigens in many organelles.