Electron microscopic immunocytochemical localization of intracellular antigens in cultured cells: the EGS and ferritin bridge procedures

Abstract

A new primary fixative, ethyldimethylaminopropyl carbodi-imide-glutaraldehyde-Tris, has been combined with the use of saponin for membrane permeabilization to yield a procedure which preserves ultrastructural morphology, yet retains a cytoplasmic matrix permeable to globulin molecules. This allows pre-embedding localization of intracellular protein antigens in cultured cells by fluorescence or electron microscopy. A further combination of these methods with the 'ferritin bridge' techniqe has allowed discrete localization which is quantifiable. Together, these methods yield an overall technique which provides high quality ultrastructural morphological preservation and precise antigen localization. Examples of the localization of alpha 2-macroglobulin, actin, SV40 T-antigen, tubulin and p60src are demonstrated. Extension of these methods from cultured cells to intact tissue should be possible without major changes.