Development of a new primary fixative for electron microscopic immunocytochemical localization of intracellular antigens in cultured cells.

We have developed a new primary fixative that permits the localization of intracellular antigens with well preserved ultrastructural morphology. This primary fixation method employs a mixture of a water soluble carbodiimide with glutaraldehyde, and preserves morphology, yet produces a permeable cytosol matrix so that antibodies can gain access to fixed proteins. Cultured cells were primarily fixed, treated with detergent to permeabilize their membranes, reacted with peroxidase labeled antibodies, secondarily fixed, and embedded in situ. The variations in morphology and accessibility of intracellular antigens were evaluated for a variety of fixatives. Concanavalin A and alpha 2 macroglobulin were chosen as examples of intracellular protein antigens to evaluate these fixation methods. Both of the proteins were localized in intracellular vesicles.