We describe a simple new technique based on the affinity of imidazole and osmium tetroxide for unsaturated lipids. Organs (e.g., kidney, liver, intestine) were perfused in vivo with a glutaraldehyde solution. Tissue fragments were then immersed in a solution containing imidazole and Os04 and are further stained with a double lead and copper citrate solution. Ultra-thin (0.06 pm) or thick (0.1-0.3 pm) sections were observed with transmission electron microscopy (80-100 kV). The method presented permits excellent visualization of cell membranes (e.g., endoplasmic reticulum,

The spatial organization of endoplasmic reticulum (ER) was examined in all segments of rat nephron. Tissues were fixed with glutaraldehyde, impregnated "en bloc" with osmium tetroxide, prepared for and examined by standard (80-100 kV) and high voltage (1 mEV) transmission electron microscopy. In all proximal tubule cells, ER forms a continuous and extensive network of canaliculi and abundant fenestrated saccules which surround mitochondria and cytoplasmic bodies; the cage-like structure of the fenestrated saccules was most evident around the spherical mitochondria of the S3 segment. In the cells of the distal straight and convoluted tubules, the network consists mostly of canaliculi with rare non-fenestrated saccules. The ER network of canaliculi is particularly rich in intercalated cells, in contrast with its rudimentary appearance in the adjacent principal cells of the collecting tubule. In fact, in these cells there are few isolated ER cisternae and they are rarely impregnated. The nuclear envelope is well impregnated in most cells throughout the various segments. Segmental variations in ER organization and its relative abundance are most likely related to the well, established functional heterogeneity of the nephron segments. Moreover, the extensive and unique organization among mitochondria, ER and the basolateral membrane suggests that these three organelles function as a unit which is related to active electrolyte transport. In addition, because of its transepithelial organization, ER may well constitute a transcellular pathway for molecules.

The endoplasmic reticulum (ER) of the columnar cells of the rat jejunum was studied with a specific block-staining technique and standard transmission electron microscopy. A new three-dimensional model, based on the analysis of stereo pair photographs, is proposed; this model suggests that the endoplasmic reticulum may constitute a transcellular route. Thick sections (0.5-1 micron) of columnar cells were made after a 5-day impregnation with osmium tetroxide and were examined by standard transmission electron microscopy at 80-100 kV. The evolution of the ER during the cellular ascent of cells from the crypt to the top of the villus is toward a greater complexity. At the base of the crypt of Lieberkühn, no definite organization is noted, and most often only the nuclear envelope and canalicular elements are stained. The endoplasmic reticulum of the mature columnar cells forms a continuous network of canaliculi and fenestrated saccules; it extends from the apex, below the microvilli, to the lateral and basal plasma membranes, and sends many projections to the nuclear envelope. In the basal part of the cell, below the nucleus, the ER consists mainly of tubular canaliculi, whereas mostly saccules are observed in the supranuclear part. The canaliculi have a diameter of 40-60 nm. Fenestrated saccules appear to form a continuous tubular structure surrounding mitochondria; the saccules have a thickness of 25-40 nm and possess irregular perforations of 35-60 nm. Finally, in some cells, the endoplasmic reticulum seems to show functional differences as reflected by the absence of reaction in cells adjacent to well-stained cells in the same part of the villus; thus the osmium impregnation technique appears to be a valid tool for studying the ER organization.

The ultrastructure of prostatic secretory cells was studied with the osmium impregnation technique in order to determine if the ER reactivity, or its absence, and its three-dimensional organization correspond to specific functions possibly hormono-dependent. Thick sections (0.3 micron) of rat ventral prostate were made after a five-day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80 kV. Studies were performed in normal adult rats, between the 3rd and 26th day following castration and in castrated rats treated with 5-alpha-dihydrotestosterone. In normal rats the impregnation technique delineated three secretory cell types (dark, greyish and clear), representing various degrees of reactivity in ER cisternae; however, despite this quantitative variation, they had similar morphological characteristics. In a longitudinal section, the ER network appeared to be made of saccules running parallel along the length of the cell and forming whorl-like patterns around the nucleus. Comparison of sections taken at various angles suggests that the ER network is made of concentric parallel saccules extending from the base to the apex of the cell and encircling the nucleus and the Golgi apparatus like a large multilayered cylinder. Whereas in dark cells the Golgi apparatus contained mostly clear vesicles, it was always heavily impregnated in clear cells. Noteworthy, osmium deposits were rarely observed on the nuclear envelope of secretory cells but were always present in basal cells. After castration, secretory cells became progressively cubic and the most conspicuous cytoplasmic change was observed in association with the ER. The Golgi apparatus decreased markedly in volume and became heavily stained with metallic osmium.(ABSTRACT TRUNCATED AT 250 WORDS)

The maturation of the endoplasmic reticulum (ER) of rat kidney tubule cells was studied with an osmium impregnation technique. Thick sections (0.3-0.6 micron) of kidney tissue were made after a five-day impregnation with osmium tetroxide and examined by standard transmission electron microscopy at 80-100 kV. Studies were performed on rat foetuses from 18-21 days of gestation, on newborns, and on 2-20 day old animals. At the undifferentiated stage, only a small percentage of the tubule cells were impregnated; in these, the perinuclear sac was stained and a few nuclear pores were already seen. Rudimentary, but thick canalicular projections seemed to originate from the perinuclear sac and become more extensive with maturity. Flattened saccules appeared later and fenestrations were seen in proximal tubule cells only when they seemed to have reached their functional specialization. In some cells, only the Golgi apparatus was stained. In the distal tubule cells, there was also progressive formation of a network consisting first of canaliculi and later of saccules which were rarely fenestrated. The osmium impregnation technique appears to be useful as an index of the ER organization development.