Immunocytochemical demonstration of LH-RH, somatostatin, and ACTH-like peptide in osmium-postfixed, resin-embedded median eminence

In guinea pig median eminences that were fixed with a glutaraldehyde paraformaldehyde mixtures, postfixed with osmium tetroxide, and embedded in araldite, immunocytochemical stainings for luteinizing hormone-releasing hormone (LH-RH), somatostatin, and alpha 17-39 ACTH have been obtained on semithin sections. These positive reactions were obtained after araldite was removed with sodium methoxide and after treatment with hydrogen peroxide. These data suggest that osmium tetraoxide postfixation can be used for the detection of low molecular weight peptides such as LH-RH, somatostatin, and ACTH-like-peptides not only in the median eminence but also in the hypothalamus.     

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.     

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.     

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 structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde

Summary Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

Fine structure of dense-cored vesicles in glomus cells of rat carotid body after fixation with permanganate or glutaraldehyde.

Glomus (Type I) cells of the carotid body of adult rats were studied electron microscopically after fixation with potassium permanganate or with glutaraldehyde and osmium tetroxide. Two permanganate fixation methods (using Krebs-Ringer-glucose, pH 7.0, or acetate buffer, pH 5.0) were compared. Numerous dense-cored vesicles were observed only in about one tenth of the glomus cells when neutral permanganate was used for fixation, although all glomus cells showed such vesicles after fixation with glutaraldehyde and osmium tetroxide. Numerous vesicles with a dense core were observed in about one third of the cells after fixation with acid potassium permanganate. With this fixation, small dense-cored vesicles similar to those in adrenergic nerve terminals were occasionally seen in the cytoplasm of glomus cells. It is tentatively concluded that the amine-storing vesicles of the carotid body are different from those in the small intensely fluorescent (SIF) cells and those in adrenergic nerve terminals.     

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.     

Application of the ferrocyanide-reduced osmium method for mineralizing cartilage: Further evidence for the enhancement of intracellular glycogen and visualization of matrix components

Summary The ferrocyanide-reduced osmium (FRO) fixation method was applied to neonatal mouse mandibular condylar cartilage for its processing for electron microscopy. The results were compared to those obtained by the conventional glutaraldehyde—osmium tetroxide fixation method. Three different stages in the life cycle of condylar cartilage cells were examined. FRO enabled the visualization of delicate fibrillar mesh in the matrix of all three zones of the cartilage, resulting in a dense appearance of the intercellular matrix. The classical stellate shape of matric granules seen in cartilage fixed with glutaraldehyde—osmium tetroxide was not observed in FRO-processed tissues. Chondrocytes that were FRO-processed almost entirely filled their lacunar space. In their pericellular area, fibrillar material and electron-dense aggregates could be demonstrated by the FRO method. As a conclusion of this study, it is recommended to supplement a conventional protocol with the FRO fixation method for routine and research purposes.     

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.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and Balbiani Rings in Chironomus salivary glands.

This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

Osmium tetroxide/p-phenylenediamine staining of nucleoli and balbiani rings in Chironomus salivary glands

Summary This paper deals with the application of the osmium tetroxide fixation followed by p-phenylenediamine treatment to salivary gland cells from Chironomus larvae. After this procedure, cytoplasm, nucleoli and Balbiani rings show a high degree of staining both in light and electron microscopy, while chromatin remains unstained. Ethanol fixation followed by osmium tetroxide/p-phenylenediamine does not modify the above mentioned staining pattern. Under these conditions, extractive procedures for lipids do not affect the osmiophilia of nucleoli and Balbiani rings, while RNase or trichloroacetic acid treatment decreaes the staining degree of these structures. In osmium tetroxide/p-phenylenediamine treated salivary glands, the highest contrast within nuclei is seen to occur in the pars granulosa from normal or segregated nucleoli, as well as in Balbiani ring granules, which appear either as hollow granules or with a bipartite or horseshoe-like structure.     

Preservation of arachidonoyl phospholipids during tissue processing for electron microscopic autoradiography

To facilitate autoradiographic subcellular localization of arachidonoyl phospholipids, the retention of radioactivity during tissue processing of murine fibrosarcoma cells labeled in vitro with 3H-arachidonate was assessed. Approximately 94% of cell radioactivity was incorporated into phospholipids. During tissue processing, extraction of radioactivity was monitored by liquid scintillation spectrometry. Fixation of cells in glutaraldehyde-tannic acid, postfixation in osmium tetroxide, en bloc staining in uranyl magnesium acetate, dehydration in ethanol, and embedding in Epon resulted in preservation of 93.5% of total tissue radioactivity. Analysis of extracted radioactivity by thin layer chromatography revealed that no specific class of phospholipids was selectively extracted. Fixation with osmium tetroxide alone was nearly as effective as the complete fixation protocol and resulted in retention of 90.0% of radioactivity. However, fixation with glutaraldehyde-tannic acid alone without osmium tetroxide post-fixation led to extraction of 69.8% of total cell radioactivity. Thus, osmium tetroxide is crucial in the preservation of arachidonoyl phospholipids and presumably forms extensive cross-links between polyunsaturated acyl residues. This degree of preservation of arachidonoyl phospholipids is indicative of spatial fixation of the radiolabeled moieties and will permit quantitative studies of subcellular loci of eicosanoid metabolism by electron microscopic autoradiography.     

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.     

Methods for the determination of adipose cell size in man and animals

Four methods for the sizing of adipose cells in small samples of human or animal adipose tissue are compared. These methods depend on the preparation of cell suspensions by incubation of the tissue with collagenase or by prolonged fixation with osmium tetroxide and separation of the fixed cells. A Coulter electronic counter was used to count and size the suspended cells and a Zeiss particle size analyzer for the sizing of cells in photomicrographs. The use of the Coulter counter to count cells in a suspension derived from a known amount of tissue and subjected to osmium tetroxide fixation is recommended for accuracy and general applicability to adipose cells of all sizes in man and animals.     

Ultrastructural localization of calcium in prothoracic gland cells of Galleria mellonella L. (Insecta,Lepidoptera) in relation to secretory activity

Summary Localization of intracellular calcium was demonstrated by precipitation with potassium hexahydroxoantimonate in the fixation medium containing osmium tetroxide or osmium tetroxide and glutaraldehyde. The presence of calcium in the precipitates was confirmed by X-ray microanalysis. Cells from active prothoracic glands contain more calcium deposits than inactive glands. The calcium precipitates are mainly localized in the nucleus, in the smooth endoplasmic reticulum, in the hyaloplasm and to a lesser degree in the mitochondria. These findings are consistent with the proposed role of calcium in the stimulation of steroidogenesis.     

Fluidity of phospholipid bilayers and membranes after exposure to osmium tetroxide and gluteraldehyde

The response of fluid bilayer regions to osmium tetroxide and glutaraldehyde fixation was examined in phospholipid multilayers and in nerve bundles from the walking legs of the lobster Homarus americanus. The samples were spinlabeled either with 5-doxylstearic acid (the 4′4′-dimethyloxazolidine-N-ozyl derivative of 5-ketostearic acid) or the maleimide spin label, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl. Osmium tetroxide fixation abolishes the characteristic orientation of the spin-labeled lipid bilayer regions and virtually eliminates motion on the electron spin resonance time scale. Glutaraldehyde treatment reduces the motion of maleimide spin labels covalently attached to proteins. However, in contrast to osmium tetroxide fixation, glutaraldehyde has essentially no effect on the orientation and mobility in the fluid bilayer regions, and hence probably does not restrict directly the potential for translational motion in membrane phospholipid bilayer regions.     

Tissue fixation and staining with osmium tetroxide: the role of phenolic compounds.

It has been postulated that phenol-containing areas of plant and animal tissues were osmiophilic, but proof of direct interaction between osmium tetroxide and phenolic materials, or the nature of such reactions, has been lacking. We find that, under conditions similar to those of normal tissue fixation, osmium tetroxide reacts rapidly with those phenols containing o-dihydroxy groups (including such species found in plant tissues) to give very stable chelate complexes. We conclude that these complexes are responsible for the observed electron-density in phenol-containing areas of tissue treated with osmium tetroxide, so that such phenols are indeed osmiophilic.     

PRESERVATION OF THE ULTRASTRUCTURE OF BACILLUS SUBTILIS BY CHEMICAL FIXATION AS VERIFIED BY FREEZE-ETCHING

The present study on the ultrastructure of Bacillus subtilis was undertaken in order to examine by means of the freeze-etching technique possible structural changes occurring during the chemical fixation procedure (Ryter-Kellenberger (R-K) fixation). Three stages were followed by freeze-etching, viz.: (a) fixation in osmium tetroxide, (b) fixation in osmium tetroxide and posttreatment with uranyl acetate, and (c) fixation in osmium tetroxide, posttreatment in uranyl acetate, and dehydration in a graded series of acetone. Preparations were made after each stage in the presence of 20% glycerol. Good preservation of ultrastructure was observed, after any of the three treatments, of the outer surface of the plasma membrane, and the inner surface of the plasma membrane. No alteration in fracturing properties could be observed. However, if we are to judge by the results of freeze-etching, any of the successive steps of the chemical fixation procedure achieve strong contrast between the nucleoplasmic region and the cytoplasm. Dependent on the quality of fixation, very delicately preserved DNA fibrils or strongly aggregated ones were seen. It appears that R-K fixation is capable of producing more or less distinctly visible changes in the native state of the nucleoplasm in young cells of B. subtilis.     

INTRAMITOCHONDRIAL FIBERS WITH DNA CHARACTERISTICS : I. Fixation and Electron Staining Reactions

Chick embryo mitochondria, studied with the electron microscope, show crista-free areas of low electron opacity. These areas are observable after fixation with osmium tetroxide, calcium permanganate, potassium permanganate, formaldehyde, acrolein, acrolein followed by osmium tetroxide, uranyl acetate followed by calcium permanganate, and acetic acid-alcohol. Staining of sections with lead hydroxide or uranyl acetate, or with both, resulted in an increased density of a fibrous material within these areas. The appearance of the fibrous structures varied with the fixative employed; after fixation with osmium tetroxide the material was clumped and bar-like (up to 400 A in diameter), whereas after treatment of osmium tetroxide-fixed tissues with uranyl acetate before dehydration the fibrous structures could be visualized as 15 to 30 A fibrils. Treatment with ethylenediaminetetraacetate (EDTA) in place of uranyl acetate coarsened the mitochondrial fibrils. After fixation with calcium permanganate or potassium permanganate, or a double fixation by uranyl acetate followed by calcium permanganate, the fibers appeared to have a pattern and ultrastructure similar to that observed after the osmium tetroxide-uranyl acetate technique, except that some of them had a slightly greater diameter (up to 50 A). Other fixatives did not preserve the fibers so well. The fibers appeared strongly clumped by formaldehyde fixation, and were difficult to identify after fixation with acrolein or acetic acid-alcohol. The staining of nucleic acid-containing structures by uranyl acetate and lead hydroxide was improved by treatment of osmium tetroxide-fixed sections with hydrogen peroxide, and the mitochondrial fibers also had an increased density in the electron beam after this procedure. The staining characteristics suggest the fibrous material of chick embryo mitochondria to be a nucleic acid-containing structure, and its variable appearance after different fixations parallels that previously reported, or described in this paper, for the nucleoplasm of bacteria and blue-green algae. The results, in addition to those described in the accompanying communication, indicate that these mitochondria contain DNA.     

Lipid fixation during preparation of chloroplasts for electron microscopy

Reaction of osmium tetroxide with isolated spinach chloroplasts fixed completely the glycolipids, phosphatidyl glycerol, and phosphatidyl choline. Under the same reaction conditions only 30% of the chlorophyll was fixed. Reaction of potassium permanganate with isolated spinach chloroplasts fixed more than 90% of the glycolipids, phosphatidyl glycerol, and phosphatidyl choline, provided the reaction period was long enough. Potassium permanganate also fixed the chlorophyll. Reaction of osmium tetroxide and potassium permanganate with isolated (14)C-lipids from Chlorella pyrenoidosa fixed 59% and 66% of the radioactivity, respectively. The lipids that were not fixed included sterols and pigments. Electron micrographs show that chloroplasts extracted with chloroform-methanol after fixation in osmium tetroxide or potassium permanganate differ from those dehydrated with acetone mainly in that in the former, osmiophilic globules have been removed and there seems to be some fusion of the boundary membranes and grana membranes. These effects may be due to the extraction of unfixed, neutral lipids such as sterols and quinones.