Osmium Tetroxide-potassium Ferrocyanide Research Articles

Glycogen distribution in the larval salivary gland cells during the development ofDrosophila melanogasterandDrosophila auraria: an ultrastructural cytochemical study

AbstractIn this study, glycogen distribution during the development of larval salivary glands in twoDrosophilaspecies (D. melanogasterandD. auraria) was examined using the osmium tetroxide–potassium ferrocyanide technique at the ultrastructural level. Glycogen particles are found in big clumps in the salivary gland cells ofD. melanogasterduring the early third instar, mainly at the basal part of the cells; they are also found as scattered particles in the cytoplasm and very close to the apical plasma membrane during the secretory activity at this developmental stage. During the middle third instar, where the production of ‘glue’ secretory granules begins, glycogen particles are mainly scattered and they are seen near the Golgi complexes and secretory granules. No glycogen particles are seen during the late third instar and secretion of ‘glue’ secretory granules. Re‐formation of glycogen begins after spiracle eversion. In the larval salivary gland cells ofD. auraria, glycogen particles (scattered and in clumps) are seen in every developmental stage. In the larval fat body cells of both species, many huge clumps of glycogen particles are seen, independent of the developmental stage. These results indicate that glycogen provides an important source of energy during development ofDrosophilasalivary glands, but different species use glycogen at different metabolic pathways, according to their needs.

Distribution of canalicular reticulum in Deiters cells and pillar cells of gerbil cochlea

Postfixation with an osmium tetroxide-potassium ferrocyanide solution revealed in supporting cells in the organ of Corti a network of canaliculi termed canalicular reticulum (CR). In Deiters cells (DCs), the CR filled cytosol at the base of the phalanx and under plasmalemma apposed to either the outer hair cells' (HCs) basal surface or nerve terminals. From these locations the CR, accompanied by dense fibrillar substance, descended along microtubule bundles and terminated by surrounding the rosette complex in the apical cytosol. Canalicular profiles protruding from the reticulum penetrated the loose meshwork comprising the periphery of the rosette complex to contact at intervals branches of the dense trabeculae that make up the core of the complex. This arrangement disclosed a structural and presumably functional relationship between outer HCs and the CR and rosette complex. Inner pillar cells (PCs) exhibited moderately abundant to sparse profiles of CR interspersed between microtubule bundles of the microtubule stalk that connected head and foot regions. More elaborate CR extended as a network upward from the top of the microtubule stalk part way into the head body and downward into a conical expansion of the stalk at the base of the cell. Cytosol on the medial side of the basal microtubule expansion contained abundant CR which in conjunction with CR between basal microtubule bundles lay situated for possible uptake of ions or neurotransmitter released from numerous adjoining nerves. CR in outer PCs resembled that in inner PCs but appeared less prevalent in the head and foot regions and did not occur in cytosol beside the basal microtubule stalk. Characteristically small Golgi complexes accompanied the reticulum in DCs and were prevalent in the upper regions but absent in the mid and lower part of inner PCs. Short cisternae in the Golgi stacks associated with CR contrasted with the lengthier cisternae in the complexes infrequently observed in cytosol outside the microtubule stalk of inner PCs.

Mitosis in normal adult guinea pig Leydig cells.

In this study, Leydig cells in mitosis in adult guinea pigs were quantified. Testes of adult control guinea pigs (n = 10) were fixed by whole body perfusion with 2.5% glutaraldehyde in cacodylate buffer, postfixed in a mixture of osmium tetroxide-potassium ferrocyanide, and embedded in Epon Araldite for qualitative and quantitative microscopy. Using stereologic techniques, the total number of Leydig cells per testis and the number of dividing Leydig cells per testis were quantified. Light microscopic studies revealed the presence of dividing Leydig cells. Ultrastructural studies on a few of these Leydig cells showed that they contained abundant smooth endoplasmic reticulum and mitochondria, which further supported this identity. The total number of Leydig cells per testis and the number of dividing Leydig cells per testis were determined as 14.1 x 10(6) (standard error [SE] = 0.33) and 8 x 10(3) (SE = 5), respectively. These results indicate that Leydig cells undergo mitosis at a rate of 1 per 1.75 x 10(4) in adult guinea pigs.

PAP labeling enhancement by osmium tetroxide-potassium ferrocyanide treatment.

We describe a procedure to intensify staining of antigens labeled by the peroxidase-antiperoxidase (PAP) method. Routinely PAP-stained samples were enhanced by application of 5% osmium tetroxide for 30 min followed by freshly prepared 2% potassium ferrocyanide, plus 1% hydrochloric acid for 15 min. The staining color changed from the original golden-brown, through transient gray, to a very dark brown that was almost black. In addition to stronger labeling, antigen locations not apparent in untreated specimens may thus be disclosed. Furthermore, retrospective staining intensification can be performed in stored PAP-labeled samples even years later.

Cytochemical identification of cerebral glycogen and glucose-6-phosphatase activity under normal and experimental conditions. II. Choroid plexus and ependymal epithelia, endothelia and pericytes.

Intracellular glycogen and glucose-6-phosphatase (G6Pase) activity were identified cytochemically within epithelia of the choroid plexus and ependyma of the cerebral ventricles including the median eminence and area postrema, the cerebral endothelium and pericytes from control, salt-stressed and fasted adult mice. Identification of glycogen was obtained by employing osmium tetroxide-potassium ferrocyanide and the periodic acid-thiocarbohydrazide-silver protein technique as ultrastructural contrast stains. A lead-capture method was used to localize G6Pase activity with glucose-6-phosphate or mannose-6-phosphate as substrate. Cerebral G6Pase functions predominantly as a phosphohydrolase to convert glucose-6-phosphate to glucose. Some glucose-6-phosphate in vivo may be derived from the breakdown of glycogen stores. Within the sampled cell types, presumptive glycogen appeared as electron-dense, isodiametric particles scattered throughout the cytoplasm. Reaction product for G6Pase activity was localized consistently within the lumen of the nuclear envelope and endoplasmic reticulum and frequently within an outer saccule of the Golgi complex under normal conditions. Choroid plexus epithelia from stressed mice exhibited a qualitative increase in cytoplasmic glycogen and a decrease in G6Pase activity; the other cell types did not express demonstrable alterations in glycogen concentration and G6Pase activity. The results indicate that glycogen and G6Pase activity are prevalent within non-neural cells of the adult mammalian CNS. Glucose utilization in the choroid plexus epithelium may be altered by stressful conditions that influence the functional activity of this cell.

Glycogen accumulations in differentiating mesonephric ducts and tubuli in male human embryos.

Human mesonephric duct epithelial cells contained empty appearing regions in the infranuclear cytoplasm when prepared for transmission electron microscopy using glutaraldehyde and osmium fixation. The same regions stained positively with PAS in Epon sections for light microscopy suggesting that glycogen was present. Incubation with saliva abolished the reaction. For electron microscopy the glycogen stained very intensely if a mixture of osmium tetroxide and potassium ferrocyanide was used instead of osmium alone. Glycogen accumulations were present between the ages of 5 to 10 weeks and absent at the age of 15 weeks. Reports by others indicate that glycogen may be present in different reactive forms in relation to its staining behaviour after various fixatives. The present results, and similar studies in other tissues, indicate that osmium tetroxide-potassium ferrocyanide fixative should be used routinely for preservation of embryos and fetuses and where indicated, for ultrastructural identification of glycogen and cytoplasmic filaments in clinical specimens.

Fine Structure and Cytochemistry of the Hydrogenosome of Tritrichomonas foetus1

Fine structural studies of the hydrogenosomes of Tritrichomonas foetus using an improved fixative reveal that they are enclosed by two closely apposed 6 nm membranes, which separate at some regions forming a large intramembranous vacuole where Ca++-binding sites are located. Fixation of the cells in a glutaraldehyde solution containing 5 mM CaCl2 and postfixation in an osmium tetroxide-potassium ferrocyanide solution led to the appearance of a reaction product associated with certain regions of the membrane of the hydrogenosomes and in the cisternae of the endoplasmic reticulum, in the recurrent flagellum, and in the plasma membrane. Treatment of ultrathin sections with EGTA removed the reaction product. These results, in association with others previously described, indicate the existence of several similarities between the hydrogenosomes and the mitochondria.

Membrane histochemistry of Physarum polycephalum myxamoebae.

Myxamoebae of Physarum polycephalum are the uninucleate, haploid stage of the organism. Histochemical studies were undertaken to characterize intracellular and plasma membranes, and to provide a basis for assaying subcellular fractions for enrichment in plasma membranes. Lead salts deposition techniques were employed for hydrolytic enzymes. Alcian blue-ruthenium red, osmium tetroxide-potassium ferrocyanide, and phosphotungstic acid-chromic acid stains were evaluated for specificity for plasma membranes. Glucose 6-phosphatase was localized in endoplasmic reticulum, Golgi apparatus, and perinuclear space. 5'-Nucleotidase was localized in food vacuoles, chromatin, and plasmalemma. Acid phosphatase was in food vacuoles and Golgi apparatus. Alkaline phosphatase was in food vacuoles and endoplasmic reticulum. We conclude that none of the above enzymes is suitable as a cytochemical marker for plasma membranes of Physarum myxamoebae, but recommend instead staining ultrathin sections of membrane pellets with phosphotungstic acid-chromic acid, which stains plasma membranes selectively.

Membrane distribution in dividing endosperm cells of Haemanthus.

Membranes in cell-wall-free dividing endosperm cells of Haemanthus were examined after postfixation with osmium tetroxide-potassium ferrocyanide. We found that preservation and staining of membranes in metaphase cells was highly variable. Even adjacent cells often showed different degrees of preservation of membrane. However, this method does reveal a much more extensive membrane system in the mitotic spindle of Haemanthus than has been revealed previously using glutaraldehyde-osmium fixation. At prometaphase a system of membranes becomes associated with the kinetochore bundles. By metaphase, membranes constitute a prominent feature of kinetochore bundles, terminating near the kinetichores. Minipoles, identified by converging microtubules and associated membranes, are distributed in a zone extending laterally across the polar regions of the cell. The microtubules appear to terminate at the minipoles, whereas the membrane system becomes oriented generally perpendicular to the spindle axis and interfaces distally with a region of amorphous electron-dense material, helical polyribosomes, and cell organelles. The role of this extensive membrane system, if any, in chromosome movement is unknown. However, its distribution is coincident with the distribution of calcium-rich membranes and kinetochore fibers at metaphase in these cells (Wolniak, S. M., P. K. Hepler, and W. T. Jackson, 1981, Eur. J. Cell Biol., 25:171-174). Thus, these membranes may function in creating calcium domains that, in turn, may play a regulatory role in chromosome movement.

The osmium tetroxide-potassium ferrocyanide (OsFeCN) staining technique for electron microscopy: a critical evaluation using ciliates, algae, mosses, and higher plants.

The osmium tetroxide-potassium ferrocyanide (OsFeCN)-method has been applied to a variety (24 objects) of ciliates, algae, mosses, and higher plants in order to test its specificity. The results showed big variation. Depending on the object, either general staining of the membranes, or selective staining of certain membranes, and/or staining of non-membraneous components in secretory vesicles and cell walls was obtained. In some cases, ultrastructural preservation was better than in controls. It is concluded that this staining technique may be helpful in tracing certain membrane systems, but it seems by no means specific for calcium-sequestering membrane systems.

The use of osmium tetroxide-potassium ferrocyanide as an extracellular tracer in electron microscopy.

Secondary treatment of glutaraldehyde fixed liver samples with long term stored solutions of osmium tetroxide and potassium ferrocyanide (Os-Fe) results in tracing of the extracellular spaces of the tissue with an electron opaque substance. This substance does not diffuse across cell membranes, its formation probably being related to the progressive reduction of the OsO4 molecule by potassium ferrocyanide. The application of the present method in electron microscopy may be useful in overcoming the artifacts often induced by other tracing techniques such as vascular perfusion with peroxidase or immersion in lanthanum. Although the period of storage of the Os-Fe solution is a clear disadvantage of the method, it seems plausible to anticipate that further studies on the chemical interaction between osmium tetroxide and potassium ferrocyanide will provide us with a Os-Fe mixture having an immediate tracer effect.