Nelson Quintana

DIFFUSION ARTIFACTS IN 3,3'-DIAMINOBENZIDINE CYTOCHEMISTRY

throughout the cytoplasm (Figs. 2 and 3). The reaction product was seen as coarse masses of granules or accumulation of fine threads forming a mesh. The albumin-containing cells were scattered diffusely throughout the lobules. Most of cells situated in the centrilobular area contained a large amount of albumin. The control sections showed no staining. The localization of albumin in liver tissue observed is similar to that reported by using an immunofluorescent antibody method (5). In the lymph nodes of rabbits hyperimmunized with HPO, the cells containing antibody against HPO were mainly localized in the medullary cords adjacent to the lumen of the lymph sinuses or in the lumen itself (Fig. 4). The antibody-containing cells were classified into two types (Fig. 5). One is plasma cell, which had an eccentrically situated nucleus and contained a large amount of antibody throughout the cytoplasm. The other cell type contained only a small amount of antibody in the thin rim of cytoplasm. These cells, round in shape and small in size, may be interpreted as lymphoblasts or lymphoplasmacytes (2). In the intercellular space, there were often small granules of the reaction product. Since the reaction product was observed only occasionally in the intercellular space in sections not exposed to HPO prior to incubation with substrate for peroxidase, most of intercellular reaction product was thought to indicate the localization of antibody in this space. There were few antibody-containing cells in the germinal center. These results are also identical with those reported with frozen or paraffin sections (9). Our results indicate that fixation, dehydration and embedding employed do not disturb the antigen-antibody reaction. Furthermore, GMA sect ions gave clearer localization of the antigen and antibody at both the cytologic and histologic level than frozen or paraffin sections. Several tissue antigens were localized immunocytochemically by Nakane (8) by using methacrylate-embedded or Epon-embedded ultrathin sections and by partially removing the embedding medium. The embedding medium is difficult to remove from thin sections, however. The use of GMA as embedding medium is expected to simplify the procedure because of the dispensability of the removal of embedding medium. REFERENCES

Pyridoxal phosphatase: cytochemical localization in GERL and other organelles of rat neurons.

A phosphatase, hydrolyzing pyridoxal-5-phosphate (P5P), a physiologically active component of the vitamin B6 complex and an essential co-enzyme in the synthesis of neurotransmitters, has been localized cytochemically in the perikarya of neurons in the peripheral, autonomic and central nervous systems of the rat. Neurons in dorsal root ganglia, sympathetic ganglia and ventral horn of spinal cord were studied by light and electron microscopy, while Purkinje cells, neurons in the dentate nucleus of the cerebellum, thalamus, and hypothalamus were studied by light microscopy only. Optimal conditions for demonstrating this activity in aldehyde-fixed tissue were determined with dorsal root ganglia. At the optimal pH of 5.0, neurons in these ganglia and in all other neurons studied show pyridoxal-5-phosphatase (P5Pase) activity in GERL. Small neurons in dorsal root ganglia also display enzyme activity in the endoplasmic reticulum (ER); activities in GERL and ER are also appreciably high at neutral pH. Small and large neurons in these ganglia, and neurons of sympathetic ganglia, show variable P5Pase activity in the Golgi apparatus. These localizations differ from the usual sites of both acid phosphatase and alkaline phosphatase activities. The P5Pase activity, demonstrated cytochemically, is a new acid hydrolase activity in GERL.

Evidence that the heads of ADH-sensitive aggrephores are clathrin-coated vesicles: Implications for aggrephore structure and function

Antidiuretic hormone (ADH) induces the fusion of long tubular organelles (aggrephores) with the luminal membrane of the receptor cell, and the delivery of particle aggregates to the membrane. Water flow is believed to take place through the particles. Nothing is known about the origin of the particle aggregates, their incorporation into the aggrephores, or the possible relationship of the aggrephores to the vesicular traffic that takes place in the epithelial cell. In the present studies of the ADH-sensitive epithelial cells of the toad urinary bladder, we have found that the spherical heads of the aggrephores appear to be clathrin-coated vesicles. We propose that vesicles originating from sites such as the Golgi or the luminal membrane may be engaged in aggrephore assembly, the resupply of particle aggregates to the aggrephores, and/or the removal of aggregates, and that the aggrephores may be central points in the pattern of vesicular traffic in the cell.

Neuronal phosphatase activities with ARA-AMP and ARA-ATP as substrates.

Phosphatases hydrolyzing adenine arabinoside-5 ‘-phosphate (ARA.AMP), an inhibitor of viral replication in the human nervous system, were studied in rat neurons. Neurons of dorsal root ganglia, fixed in glutaraldehyde and formaldehyde, were studied by light and electron microscopy, while neurons of sympathetic ganglia, thalamus, hypothalamus, cerebral cortex, and spinal cord were observed by light microscopy only. Localizations of phosphatases hydrolyzing ARA.AMP differed from acid phosphatase (AcPase) localizations when cytidine-5’-phosphate (CMP) was used as substrate. However, localizations paralleled those described earlier for pyridoxal-5’-phosphatase (P5Pase): electron microscopy of small and large neurons in dorsal root ganglia showed the most consistent activity in GERL. Both the distribution and intensity of reaction in elements of the Golgi apparatus were variable. Reaction product was also present in the endoplasmic re-