Adaptation of the reverse hemolytic plaque assay to electron microscopy: a study of the individual secretory activity in prolactin cell subpopulations.

Abstract

We used the reverse hemolytic plaque assay (RHPA), a method for detecting secretion by single cells, to demonstrate functional heterogeneity among prolactin (PRL) cells of rat anterior pituitary at the light microscopic (LM) level. We attempted to adapt RHPA for electron microscopy (EM) to define the relationships between fine structure and secretory activity in individual PRL cells. A major modification of the technique, intended to improve preservation of ultrastructure, was allowing the cells to recover after their enzymatic dispersion from the pituitary tissue, through a brief (3-4 hr) culture step before the assay. Adaptation for EM was achieved by the use of plastic slides for construction of specialized Cunningham chambers, permitting application of all the EM procedures (flat embedding, punching of selected areas) usually employed for cultured pituitary monolayers. Moreover, immunocytochemical pre- and post-embedding methods were also applied for cell identification and study of subcellular hormone distribution. Such a modified RHPA enabled us to analyze the ultrastructure of plaque-forming cells surrounded by their companion red blood cell ghosts. The first results with EM RHPA showed that under basal conditions a subpopulation of PRL cells containing small granules (150-200 nm) was actively secreting, whereas PRL cells with large (300-600 nm) and irregular granules appeared to be stimulated by thyrotropin-releasing hormone or KCl. The EM RHPA technique described here might receive more general application and could be utilized for study of many other secretory systems.