THE EFFECTS OF RESERPINE AND HYPOXIA ON THE AMINE-STORING GRANULES OF THE HAMSTER CAROTID BODY

The carotid bodies from control, reserpine-treated, and hypoxia-treated hamsters were fixed with phosphate-buffered glutaraldehyde and osmium tetroxide, s-Collidine-buffered osmium tetroxide, or phosphate-buffered glutaraldehyde followed by potassium dichromate incubation. Following glutaraldehyde-osmium tetroxide fixation no differences in density or population of the electron-opaque granules in the glomus cells of either control or experimental animals were observed. With s-Collidine-buffered osmium tetroxide and the glutaraldehyde-dichromate technique a marked decrease in density without an appreciable reduction in number of granules was noted after reserpine treatment, while in hypoxia-treated hamsters the density and population of the granules were not different from those of the controls. The results indicate that reserpine depletes the amines without granule disappearance and that hypoxia does not affect the amine content of the granules. It is suggested that following glutaraldehyde-osmium tetroxide double fixation, persistence of the density of the granules in reserpine-treated animals is due primarily to the nonamine content, and that the amines in the glomus cells are probably not directly involved in the respiratory reflex.     

Quantitation of ultrastructural changes in mouse pancreatic acinar cells caused by foreign serum

Quantitative changes in the pancreatic acinar cell organelles were studied in BALB/c mice injected with 1.0 ml fresh rabbit serum intraperitoneally. Groups of 5 mice were killed at 0, 1, 3, 6 and 12 h after the serum injection. Pancreatic tissue was processed for electron microscopy by glutaraldehyde and osmium tetroxide fixation and Epon embedding. The proportions of acinar cell cytoplasm (volume fractions) occupied by zymogen granules, granular endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes (including autophagosomes) were determined by the point counting method from electron micrographs. The volume fraction of lysosomes increased during the first 3 h and remained markedly elevated up to 12 h. The volume fractions of zymogen granules increased from 12 to 28% in 12 h. It was concluded that the secretory mechanism of pancreatic acinar cells was injured by the foreign serum. The injury caused accumulation of zymogen granules and increased autophagic activity in the acinar cells.     

The effects of 6-hydroxydopamine on the appearance of granulated vesicles in glomus cells of the rat carotid body

The glomus cells of the rat carotid body reveal an intense fluorescence after exposure to paraformaldehyde vapor and contain catecholamines. After initial fixation in glutaraldehyde, many granulated vesicles are seen in the glomus cells. After initial fixation in osmium tetroxide, most of the vesicles are depleted of their dense interiors and granulated vesicles occur infrequently. Administration of 6-hydroxydopamine followed by initial fixation in osmium tetroxide leads to the reappearance of dense interiors in virtually all vesicles. 6-Hydroxydopamine apparently is taken up by the membrane pump of the glomus cell and is incorporated into the amine storage granules, thereby displacing the endogenous monoamines. Osmium tetroxide does not dissolve the 6-hydroxydopamine from the vesicles, as it apparently does for the normal vesicular contents. The 6-hydroxydopamine does not fluoresce, hence 6-hydroxydopamine administration results in a decreased intensity of formaldehyde induced fluorescence in the glomus cells. Administration of reserpine after 6-hydroxydopamine treatment (and subsequent initial fixation in osmium tetroxide) depletes the previously restored dense material from the vesicles of the glomus cells. 6-Hydroxydopamine acts like a monoamine in that it is taken up by the glomus cell, incorporated into the vesicles, and can be depleted from the vesicles by reserpine.     

The reactions between formaldehyde,glutaraldehyde and osmium tetroxide,and their fixation effects on bovine serum albumin and on tissue blocks

Summary The reactions between osmium tetroxide and glutaraldehyde and formaldehyde were investigated. It was found that they react together to form intermediate products which then break down to form osmium black. Glutaraldehyde reacts much more rapidly with osmium tetroxide than formaldehyde. The rates of the reactions are increased by increasing the glutaraldehyde concentration or adding bovine serum albumin to the reaction mixture. The reaction rates increase with temperature. The mixtures of fixatives were also tried on tissues and the results paralleled the model experiments. The crosslinking of bovine serum albumin by osmium tetroxide, formaldehyde and glutaraldehyde singly and in mixtures was quantitatively assessed by viscosimetry, gel filtration and disc electrophoresis coupled with densitometry. The crosslinking of bovine serum albumin by pairs of fixatives was less than that produced by the most effective of the pair. After 5 min reaction osmium tetroxide was the most effective crosslinking agent according to viscosimetric experiments, but after one hour's reaction with bovine serum albumin, glutaraldehyde was revealed as the most effective crosslinking agent by gel filtration and electrophoresis.     

An improved immunocytochemical method for subcellular localization of serotonin in rat enterochromaffin cells

Serotonin-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of 5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and proximal colon, fixed in glutaraldehyde only. In all three locations, the majority of the gold particles (90%) in EC cells were localized over the dense core of the secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HT-LI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules.     

Electron microscopic localization of chromogranin A in osmium-fixed neuroendocrine cells with a protein A-gold technique

An antibody (LK2H10) to chromogranin A has been recommended for use in ultrastructural identification of neuroendocrine secretory granules. Previous studies have demonstrated immunoreactive chromogranin A in specimens prepared for electron microscopy by glutaraldehyde fixation only. In this study, the effect of specimen post-fixation by osmium tetroxide on post-embedding localization of chromogranin A was evaluated. Human tissues from benign endocrine glands, neuroendocrine tumors, and non-neuroendocrine tumors were post-fixed in osmium, embedded in epoxy resin, and the sample thin sections immunolabeled using a protein A-gold technique. Chromogranin A-positive neurosecretory granules were detected in pancreatic islets, adrenal medulla, stomach, ileum, anterior pituitary, and parathyroid. Mid-gut carcinoids, bronchial carcinoids, pheochromocytomas, paragangliomas, carotid body tumors, and thyroid medullary carcinomas contained immunoreactive granules. Cytoplasmic granules in non-neuroendocrine tumors did not react for chromogranin A. Tissues post-fixed in osmium tetroxide had optimally preserved ultrastructural features, and use of this fixative is compatible with postembedding localization of chromogranin A in neurosecretory granules.     

Ultrastructure of the concentric membrane system in Arthrinium aureum

An ultrastructural study was performed on Arthrinium aureum. The fungi were treated with glutaraldehyde and osmium tetroxide fixation. The hypha and conidia has a concentric membrane system which consisted of multiple membranes of a myelinoid appearance, and continued to the conidia and hypha plasma membrane. The fungi were also treated with periodic acid-alkaline bismuth (PABi) staining after glutaraldehyde and osmium tetroxide fixation. PABi positive materials were found on the marginal glycogen granules, the concentric membrane system and the conidia plasma membrane.     

The effects of various fixatives on the relative thickness of cellular membranes in the ventral lobe of the rat prostate

Synopsis A densitometric method was utilized in the measurement of the relative thickness of the cellular membranes in the ventral lobe of the rat prostate. Potassium permanganate, glutaraldehyde, osmium tetroxide, and ruthenium tetroxide solutions were used as fixatives. During preparation for electron microscopy, the tissues were given standardized treatments to reduce methodological errors; latex particles were applied to the thin sections to serve as reference particles of a known size. The most remarkable observation of the study was that the densitometric method yielded reproducible results and that the different fixatives gave significantly different values for the relative thickness of cellular membranes. Glutaraldehyde, or glutaraldehyde followed by ruthenium tetroxide post-fixation, gave the highest values for membrane thickness while osmium tetroxide and potassium permanganate gave the lowest values. Glutaraldehyde treatment, prior to osmium tetroxide or potassium permanganate post-fixations, rendered the membranes thicker than after osmium tetroxide and potassium permanganate treatments alone. Ruthenium tetroxide appeared to be very suitable for fixation of cellular membranes.     

FINE STRUCTURAL CHANGES IN RESPONSE TO HORMONAL STIMULATION OF THE PERFUSED CANINE PANCREAS

The dog pancreas isolated in situ was perfused with oxygenated dog blood and stimulated with pancreozymin, secretin, or both. There were no significant changes in the fine structure of the acinar, centroacinar, or duct cells attributable to the perfusion. Combined glutaraldehyde and osmium fixation gave good preservation of the secretory products of the acinar cell. Before stimulation, the lumen of the acini is filled with material similar in texture to the content of the zymogen granules, but of somewhat lower density. Release of secretion commonly takes place by coalescence of the limiting membrane of zymogen granules with the plasmalemma, but one granule opening at the surface may frequently be joined by others coalescing with its membrane and forming an interconnected series all with contents having the same texture as the released zymogen. Such a mechanism seems to permit a more rapid release of secretory product than discharge of individual granules. Pancreozymin stimulation caused marked depletion of zymogen granules, but no obvious changes in the Golgi apparatus. It is clear, therefore, that this hormone exerts its effect upon release of granules rather than upon their formation. Secretin stimulation of water and bicarbonate secretion caused a marked washing out of the luminal contents, but had little detectable effect on cellular structure.     

ULTRASTRUCTURAL LOCALIZATION OF DIALYZED IRON-REACTIVE MUCOSUBSTANCE IN RABBIT HETEROPHILS, BASOPHILS, AND EOSINOPHILS

For ultrastructural localization of acid mucosubstances in rabbit granulocytes, bone marrow and buffy coat specimens were fixed with formalin, glutaraldehyde, or osmium tetroxide, sectioned at 40 µ, and stained with the Rinehart and Abul-Haj solution of dialyzed iron (DI). Heterophils revealed DI staining on the outer surface of the plasma membrane, in the Golgi complex involved in primary granulogenesis, and in primary granules. The intragranular distribution of DI-stained material varied at different stages in the maturation of primary granules. Immature granules of heterophils fixed by any of the three methods contained a peripheral concentric band of DI-positive material; however, fully mature primary granules possessed a core of DI-reactive material in heterophils fixed with osmium tetroxide, but they contained little or no staining in heterophils fixed with formalin or glutaraldehyde. Secondary granules of rabbit heterophils failed to stain with DI. Tertiary granules, observed only in late heterophils, contained distinct DI-positive particles. Basophil granules exhibited intensely DI-stained material distributed in an orderly pattern throughout the granule. In eosinophils, DI staining was localized in the Golgi complex and in the rims of a few immature cytoplasmic granules.     

Ultrastructural cytochemistry of the secretory granules of the hamster submandibular gland

Summary The ultrastructure and cytochemistry of the secretory granules of the male hamster submandibular salivary gland were studied. After fixation in glutaraldehyde followed by osmium tetroxide the granules exhibit a characteristic bipartite substructure, with an electron lucid crescenteric rim and a more dense central core. A differentiation into two regions of the granules could also be visualized in specimens primarily fixed in Millonig's osmium tetroxide or in potassium permanganate. The electron lucid peripheral portion of the membrane bounded secretory granules further displays a strong positive reaction after staining of ultrathin sections with the periodic acid-chromic acid-(PA-CrA)-silver technique. The strong periodate reactivity of the rim relative to the core, suggests a difference in mucin composition of the two granule regions. With the PA-CrA-silver staining technique a positive reaction was also observed within the Golgi apparatus of the acinar cells.     

THE STAINING OF THIN SECTIONS OF MOUSE PANCREAS PREPARED BY THE FERNÁNDEZ-MORÁN HELIUM II FREEZE-SUBSTITUTION METHOD

Small pieces of mouse pancreas were rapidly frozen in helium II, substituted in methanol at -75°C., and embedded in methacrylate by ultraviolet polymerization in the cold. The unstained cells show a structure similar to that after OsO₄ fixation, except that the RNP particles have little or no contrast and the mitochondria and Golgi zones appear as grey areas without internal structure. After staining the sections by floating them on solutions of lead acetate or osmium tetroxide, there is an increase in contrast of RNP particles, ergastoplasmic membranes, and zymogen granules. Mitochondrial and Golgi membranes, zymogen granule membranes, and a membrane along the outside of the ergastoplasmic cisterna appear in negative contrast. The structure of the ergastoplasm, the existence of RNP particles, and the production of negative contrast are discussed. A modification of Gomori's method for acid phosphatase produces a lead deposit around the periphery of the zymogen granules. Possibly this deposit does not represent the true site of the enzyme, but the results show the feasibility of histochemistry at the level of resolution of the electron microscope.     

Ultrastructural localization of immunoreactive neurophysins using monoclonal antibodies and protein A-gold

Using three different monoclonal antibodies against rat neurophysins (5), with protein A-gold as immunocytochemical marker (27), the murid hypothalamoneurohy-pophysial system was studied at the ultrastructural level. Postembedding staining was done on epoxy-embedded sections of supraoptic nuclei and posterior pituitaries. Specific immunolabeling of vasopressinergic and oxytocinergic neurosecretory granules was observed in tissues fixed with glutaraldehyde or glutaraldehyde mixtures (containing paraformaldehyde and picric acid), with or without osmium tetroxide postfixation and with or without sodium metaperiodate oxidation. Some autophagic vacuoles containing lysed neurosecretory granules were also neurophysin immunoreactive. Nonspecific background staining was extremely low. An attempt was made to appraise labeling intensities semiquantitatively by counting gold particles in relation to number of secretory granules per axonal varicosity. Immunoreactivity was measurably influenced by the mode of fixation, sodium metaperiodate oxidation, and titer and affinity of the antibody. The protein A-gold technique using monoclonal antibodies against neurophysins provides a superior means of ultrastructural analysis of the hypothalamoneurohypophysial system, both visually and morphometrically.     

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow.

The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.     

Schistosoma mansoni: a new parenchymal cell in cercariae

A new type of cell has been identified in cercariae of Schistosoma mansoni. The perikarya (cell bodies) of these cells were located in the body (midsegment), in an area oral to the acetabulum (ventral sucker). Cytoplasmic processes extending from the perikarya ramified throughout the parenchyma of the anterior organ (oral sucker), body, and tail segments by following the path of the nerve processes from the neuropile. The perikarya of these cells had heterochromatic nuclei and a predominance of particulate material and granules (240-360 nm) in their cytoplasm. Aggregates of granules (240-360 nm) and associated vesicles (34 nm) were scattered throughout the cytoplasmic processes of the cells and formed distinct varicosed areas. These processes often connected to the tegument in the midsegment (body) of the cercariae. The granules and associated vesicles reacted (became electron dense) with fixatives reported to be detectors of biogenic amines: The glutaraldehyde/osmium tetroxide fixation procedure rendered the granules electron dense while the glutaraldehyde/chromate/osmium tetroxide fixation procedure rendered the granules and the associated vesicles electron dense. The chromate solution of the latter procedure was responsible for the electron density of the associated vesicles. The morphology of these cells (their long ramifying cytoplasmic processes) and their reaction to chromium suggests that they are probably biogenic aminergic sensory cells.     

Imidazole-buffered osmium tetroxide: an excellent stain for visualization of lipids in transmission electron microscopy

Summary The usefulness of imidazole-buffered osmium tetroxide as a stain for lipids in transmission electron microscopy has been investigated. Rat liver and other tissues were fixed by perfusion with glutaraldehyde and post-fixed with osmium-imidazole and the appearance of lipid droplets was compared with that after post-fixation in unbuffered aqueous osmium tetroxide or an osmium solution buffered otherwise. Prominent electron-opaque staining of lipid droplets and of lipoprotein particles was noted after post-fixation with 2% osmium-imidazole, pH 7.5, for 30 min. The lipid droplets appeared well circumscribed with no evidence of diffusion. In contrast, the intensity of staining was much less and there was some diffusion around lipid droplets in material post-fixed in aqueous or cacodylate-buffered osmium tetroxide. Spot tests on filter paper revealed that unsaturated fatty acids, especially linolenic and linoleic acids reacted more intensely with osmium-imidazole than with aqueous osmium tetroxide. These findings demonstrate that osmium-imidazole provides an excellent stain for lipids in transmission electron microscopy and that most probably it stains lipids with unsaturated fatty acids.     

Swelling of golgi apparatus cisternae in monensin-treated tissues is modulated by fixation conditions

Summary Swelling of Golgi apparatus cisternae is reported to be a common response to the ionophore, monensin. However, the amount of swelling depends on fixation, thus raising the question of whether the swelling response is due to monensin or to the fixation protocol. To resolve this problem, maize root cap cells were treated with monensin and then fixed with glutaraldehyde and osmium tetroxide (applied sequentially), osmium tetroxide alone, or aqueous potassium permanganate, or were quick frozen in liquid propane and substituted in acetone-osmium tetroxide. The chemical fixatives (which take minutes to stabilize tissue elements) were judged by comparison with freeze substitution which requires only fractions of a second to stabilize tissue elements. The results verify that monensin causes cisternal swelling and that this swelling is best observed at the ultrastructural level by fixation in glutaraldehyde/osmium tetroxide or by freeze substitution.     

Application of silver stains to gastric endocrine cells in plastic sections

Summary A simultaneous light and electron microscopic study of mouse gastric mucosa was made to determine whether the silver nitrate methenamine stain of Duk-Ho Lee could be used to stain gastric endocrine-like cells in plastic embedded tissue. Examination of consecutive thick and thin sections showed that this stain blackened the granules of the predominant type of endocrine-like cell present. Blackening of the granules with silver occured in tissue fixed in osmium tetroxide solution with or without dichromate salt or in tissue fixed in glutaraldehyde then treated with osmium. The intensity of staining was deepest in the osmium-dichromate fixed tissue, but the glutaraldehyde-osmium procedure gave less interference from diffuse silver impregnation and better preservation of detail for electron microscopy.     

Structure of the nucleoid in cells of Streptococcus faecalis

The structure of the nucleoid of Streptococcus faecalis (ATCC 9790) was examined and compared in the unfixed and fixed states by immersive refractometry and electron microscopy. It appears from these studies that the nucleoid structure is much more centralized in unfixed chloramphenicol-treated (stationary-phase) cells than it is in cells in the exponential phase of growth. The more dispersed configuration of the exponential-phase nucleoid could be preserved by fixation in glutaraldehyde, but not in Formalin or in osmium tetroxide. One important factor in explaining these differences in preservation is that glutaraldehyde (but not Formalin or osmium tetroxide) can rapidly cross-link the amino groups of macromolecules in cells. It was also observed that osmium tetroxide resulted in a preferential breakdown of nascent ribonucleic acid. These results are interpreted as indicating that glutaraldehyde is able to stabilize the exponential-phase nucleoid before it assumes the more central appearance seen in osmium tetroxide- and Formalin-fixed cells. These results are discussed in terms of the proposed organization of the exponential-phase nucleoid in unfixed cells.     

HISTOLOGICAL STAINING OF LIPIDS FOR THE LIGHT AND ELECTRON MICROSCOPE

1. It is generally agreed that the blackening of osmium tetroxide by unsaturated lipid is too unpredictable to demonstrate lipid in tissues.
2. At neutral pH osmium tetroxide combines with the double bonds in the lipoproteins of cellular membranes (mitochondria, etc.) and the deep colour reaction of ethyl gallate with this osmium provides good staining of lipid for the light microscope.
3. Osmium taken up by tissue proteins at neutral pH is only a small fraction of that taken up by the lipid. (After acid fixatives osmium tetroxide is a general protein stain.)
4. The uptake of Sudan black B by partition from dilute solution is a specific test for lipid, but in normally fixed tissue most of the structural lipid is 'bound' and is not accessible to the dye.
5. Cautious treatment of fixed tissue with dilute sodium hypochlorite will unmask this lipid for viewing by the light microscope.
6. Direct fixation with neutral osmium tetroxide is an effective method for visualizing lipid for the electron microscope (as in the ethyl gallate method for the light microscope). But the poor penetration of osmium limits its use in this way.
7. After formol/glutaraldehyde fixation much of the lipid in the tissues is 'bound' and does not take up osmium. It can be unmasked by a saturated aqueous solution of thymol.
8. The unmasked lipid can then be rendered more osmiophil by partition in a solution of the highly unsaturated terpene farnesol, thus increasing the uptake of osmium in a renewed application.
9. Some of the novel observations on tissue lipids made by these methods are reviewed.