Vila-Porcile E, Picart R, Tougard C
Collège de France, Groupe de Biologie de la Cellule Neuroendocrine, URA CNRS D 1115, Paris, France.
J Histochem Cytochem. 1994 Jan;42(1):11-22. doi: 10.1177/42.1.8263322.
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.
我们使用反向溶血空斑试验（RHPA），一种检测单细胞分泌的方法，在光学显微镜（LM）水平上证明大鼠垂体前叶催乳素（PRL）细胞之间的功能异质性。我们试图将RHPA应用于电子显微镜（EM），以确定单个PRL细胞的精细结构与分泌活性之间的关系。该技术的一个主要改进是，为了改善超微结构的保存，在检测前通过一个短暂的（3-4小时）培养步骤，使细胞在从垂体组织酶分散后恢复。通过使用塑料载玻片构建专门的坎宁安室实现了对EM的适应性，允许应用通常用于培养垂体单层的所有EM程序（平板包埋、选定区域打孔）。此外，免疫细胞化学的包埋前和包埋后方法也用于细胞鉴定和亚细胞激素分布的研究。这种改良的RHPA使我们能够分析被其伴随的红细胞幽灵包围的形成空斑细胞的超微结构。EM RHPA的初步结果表明，在基础条件下，含有小颗粒（150-200纳米）的PRL细胞亚群在活跃分泌，而含有大颗粒（300-600纳米）和不规则颗粒的PRL细胞似乎受到促甲状腺激素释放激素或氯化钾的刺激。这里描述的EM RHPA技术可能会得到更广泛的应用，并可用于研究许多其他分泌系统。
