A fixation method for improved antibody penetration in electron microscopical immunoperoxidase studies

Abstract

A fixation method for electron microscopical immunoperoxidase staining has been developed, which (a) allows penetration of antibodies through cell membranes to intracellular antigen sites, (b) provides a reasonable cell preservation and (c) does not alter the antigenic structure in too great an extent. Penetration of the antibodies has been achieved by using saponin as a cell membrane attacking agent. The best results could be obtained after pretreatment of cell monolayers with a mixture of 0.05% saponin, 0.0125%-0.05% glutaraldehyde and 1% paraformaldehyde for 5 min at 4 degrees C, and postfixing them with the corresponding fixative without saponin for 45 min at 4 degrees C.