THREE-DIMENSIONAL OBSERVATIONS ON THICK BIOLOGICAL SPECIMENS BY HIGH VOLTAGE ELECTRON MICROSCOPY

Thick biological specimens prepared as whole mount cultured cells or thick sections from embedded tissues were stained with histochemical reactions, such as thiamine pyrophosphatase, glucose-6-phosphatase, cytochrome oxidase, acid phosphatase, DAB reactions and radioautography, to observe 3-D ultrastructures of cell organelles producing stereo-pairs by high voltage electron microscopy at accerelating voltages of 400-1000 kV. The organelles demonstrated were Golgi apparatus, endoplasmic reticulum, mitochondria, lysosomes, peroxisomes, pinocytotic vesicles and incorporations of radioactive compounds. As the results, those cell organelles were observed 3- dimensionally and the relative relationships between these organelles were demonstrated.

Improved method for post-embedding cytochemistry using reduced osmium and LR white resin.

We established an improved method for post-embedding cytochemistry by which highly specific cytochemical reactions on excellent cellular ultrastructures are possible. The method is a combination of post-fixation in potassium ferrocyanide-reduced OsO4 and embedment in acrylic-based LR White resin. It permits both immuno- and lectin-gold cytochemistry with fine ultrastructures comparable to those obtained by conventionally osmicated and epoxy-embedded tissues. Fixation with reduced osmium appeared to contribute to the preservation of immunoreactivity and membranous structures. By this method, the immunocytochemical localization of secretory proteins (amylase and chymotrypsinogen), actin filaments by polyclonal antibodies, 105 KD Golgi-associated protein by a monoclonal antibody (GF-1), and binding sites for gold-labeled lectin could be demonstrated. Also possible was multiple staining with enzyme cytochemistry of thiamine pyrophosphatase and immunocytochemistry of GF-1 and anti-amylase antibodies. This multiple staining made possible partial characterization of the trans-Golgi network in parotid acinar cells.