Application of confocal laser scanning microscopy to the deep pineal gland and other neural tissues

The study of the deep pineal gland of the Mongolian gerbil and other neuronal tissue from the rat by means of confocal laser scanning microscopy (CLSM) is described. Opical serial sectioning was performed on thick (100–200 μm) sections of the deep pineal gland of the Mongolian gerbil stained immunohis‐tochemically using antisera to S‐antigen and tyrosine hydroxylase (TH). Both dual‐stained and single‐stained material was examined using the fluorochromes fluorescein isothiocyanate (FITC) and Texas Red. High resolution images were obtained showing that pinealocytes have 1–3 processes that extend primarily to other pinealocytes or presumptive pinealocytes. Pinealocytes are located within the deep pineal gland as well as adjacent to the posterior aspect of the medial habenular nuclei. Pinealocyte processes were not seen extending into the habenular nuclei, but rather ended within the deep pineal gland a significant distance from their perikarya. The TH‐immunopositive fibers were distributed throughout the deep pineal gland, often forming “baskets” of fibers around pinealocytes rather than being associated primarily with blood vessels. Other uses of the confocal microscope are demonstrated on rat neural tissue reacted with peroxidase/diaminobenzidine (DAB) immunohistochemistry and FITC fluorescence immunohistochemistry (paraventricular nucleus) as well as Golgi‐stained neuronal tissue (cerebral cortex). The HRP/DAB and Golgi‐stained images were visualized using the reflected image mode of the confocal system. A summary of the advantages of CLSM include the following: (1) the optical sectioning capabilities allow for true three‐dimensional imaging thus aiding in the determination of the three‐dimensional structure of a cell plus cell‐cell associations, (2) intact tissue as well as thick sections can be used without mechanical sectioning artifacts, (3) higher resolution can be obtained due to elimination of out‐of‐focus fluorescence, (4) CLSM requires significantly less time than other three‐dimensional reconstruction techniques, and (5) once computerized, the data can be analyzed or processed in a variety of ways.