Freeze-fracture immunocytochemistry for intracellular localization of serotonin in mast cells stimulated with compound 48/80

Abstract

Changes of intracellular localization of serotonin in rat mast cells were examined by freeze-fracture immunocytochemistry, to prevent the translocation of the serotonin antigen. Rat peritoneal cells including mast cells were stimulated in vitro with compound 48/80, at 17 degrees C for 0, 30 or 60 s for exocytosis to occur. The mast cells were fixed, quickly frozen and freeze-fractured to expose the antigen on the fractured surface. They were immunostained with serotonin antibody, and the immunoreactions on the fractured surface were examined on ultrathin sections by electron microscopy. Unstimulated mast cells exhibited serotonin localization mostly in each intragranular matrix. In contrast, mast cells stimulated for 30 s exhibited increased serotonin in their intergranular cytoplasm. Mast cells showed more distinct immunoreactions in the cytoplasm where degranulation would be promoted after 60 s. It is suggested that intracellular release of serotonin occurred in the stimulated mast cells.