Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

Ultrastructure of complex carbohydrates of rodent and monkey ependymal glycocalyx and meninges

The surfaces of the brain offer metabolic and mechanical support to the underlying parenchyma. Mouse, rat, and monkey brains were fixed by immersion in a glutaraldehyde fixative or glutaraldehyde with cetylpyridinium chloride, followed by block staining for complex carbohydrates using alcian blue with OsO4 postfixation, or OsO4 postfixative solution containing ruthenium red, or alcian blue and then ruthenium red-OsO4 treatment. The ependyma in these species had a glycocalyx extending into the ventricular fluid as a finely filamentous network when stained with alcian blue or with alcian blue followed by ruthenium red-OsO4. Mice in the middle age range had stained material in this glycocalyx resembling the hyaluronic acid reported in the ocular vitreous body. Similar material was seen in the arachnoidal space of these mice and in the inner connective tissue matrix of the dura mater. Both the mouse and monkey had a cell-free zone, termed the inner dural matrix zone, between the thick fibrous dura and its innermost cellular layer. This zone contained filamentous and globular alcian blue-stained material. The complex carbohydrates of the mouse ependymal glycocalyx and inner dural matrix zone underwent changes developmentally. Aged rats were injected intraventricularly with latex beads, which, along with extravasated erythrocytes, were seen to adhere to the ependymal glycocalyx. A similar adhesion of erythrocytes was seen in the mouse and monkey ependymal glycocalyx and in the filamentous network of the mouse and monkey inner dural matrix zone. The ependymal glycocalyx, formed in part of complex carbohydrates, is much thicker than previously demonstrated. Some activities related to the ependymal lining of the ventricles, including the movement of cells or particles, the penetration of metabolites or serum-protein fractions (e.g., immunoglobulins), and cell-surface hydration, probably depend in part on complex carbohydrates that provide a sticky, electrically negative, hydrophilic environment. The complex carbohydrates in the inner dural matrix zone might provide mechanical buffering. Complex carbohydrates in the arachnoidal space may help to maintain a loose tissue that needs not only to be hydrated, but also to be open enough to provide cerebrospinal fluid circulation.     

Increase in the calcium content of cardiac tissue after postfixation with osmium tetroxide

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z2/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium treated than in nontreated tissue. However, this result is observed with OsO4 solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO4 solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO4 fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.     

Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue

Cardiac conduction fibers fixed either in glutaraldehyde and OsO4 or treated additionally en bloc with uranyl acetate were studied in order to demonstrate the structure of glycosomes (protein-glycogen complex). Sections were stained histochemically by periodic acid-thiosemicarbazide-silver proteinate (PA--TSC--SP) for glycogen followed by uranyl acetate and lead citrate (U-Pb) for protein. In control sections periodic acid was replaced by hydrogen peroxide (H2O2). Glycogen appeared in all sections stained by PA-TSC-SP. Protein was poorly contrasted in periodic acid treated histochemical sections taken from fixed in glutaraldehyde and OsO4. Simultaneous staining of glycogen and protein was achieved in sections of tissue treated en bloc with uranyl acetate. This treatment revealed two classes of glycosomes: 1) glycosomes deposited freely in the cytoplasm whose structure was disintegrated after treatment with uranyl acetate: 2) glycosomes associated with other cellular structures that remained intact. Staining of glycogen and protein in the same section demonstrated for the first time the structure of intact glycosomes.     

An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections.

Immunoelectron microscopy using chromogranin A-specific antibodies has been proposed as an efficient technique for identification of secretory granules (SGs) in tumor cells with evidence of apparent neuroendocrine differentiation. Using an antigen retrieval (AR) method, we succeeded in immunolabeling SGs with antibodies in ultrathin sections of routinely processed epoxy-embedded blocks of tissue. Samples of an insulinoma were fixed in 2% glutaraldehyde, postfixed in 1% OsO(4), and embedded in epoxy resin. Ultrathin sections were immunostained with chromogranin A-specific antibodies and gold-conjugated second antibodies. There was no significant labeling in the absence of AR. Neither etching with sodium metaperiodate nor microwave irradiation of ultrathin sections in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0) was effective in improving the efficiency of immunolabeling. However, ultrathin epoxy-embedded sections that were microwaved in alkaline solution (pH 10) were adequately labeled (5.2 +/- 0.34 particles per SG). Moreover, considerably improved efficiency of immunostaining was achieved by microwaving sections in alkaline solution (pH 10) with subsequent immunostaining at 60C (12.2 +/- 0.51 particles per SG). This method can also be applied to epoxy-embedded sections obtained from formalin-fixed, paraffin-embedded blocks of tissue and was even valid for an old epoxy-embedded block of tissue prepared 15 years previously.     

Observations on the Kinetics of Uranyl Acetate and Phosphotungstic Acid Staining of Chromatin in thin Sections for Electron Microscopy

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO₄) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with gutaraldehyde only, but stain uptake is reduced following a long wash with distilled water to a level similar to that seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO₄ increases UAc uptake by a factor of about 3.

The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO₄; similar levels for final PTA uptake are also observed.

An increase in the resin content of embedded chromatin postfixed with OsO₄ is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.     

Electron microscopical demonstration of polysaccharides in rotifers (Aschelminthes) with the use of OsO4-ferricyanide (author's transl)]

Three species of rotifers were fixed with glutaraldehyde and postfixed in a OsO4-ferricyanide solution. This procedure results in staining of the sarcoplasmic reticulum (SR) and the surface coat of muscle cells as well as in staining of the surface coat of glands and nerve cells. Section-staining with leadcitrate increased the electron opacity of the precipitates, which are interpreted as polysaccharides. The partial decrease of staining of polysaccharides, which sometimes is observed, may be related to processes of function. The precipitates within the SR are considered as glycogen-beta-particles.     

Electron microscopic localization of concanavalin A receptor sites in pollen surface material after fixation with glutaraldehyde-cetylpyridinium chloride

Pollen from birch trees (Betula pendula) was fixed in glutaraldehyde containing 0.5% cetylpyridinium chloride (CPC), incubated with concanavalin A (Con A)-ferritin, postfixed in osmium, dehydrated, and embedded in Epon. On ultrathin sections, ferritin particles were observed closely associated with the electron-dense material precipitated by CPC on the surface of the pollen grains. Controls for CPC, which were fixed in glutaraldehyde alone, showed no electron-dense material on the surface. In controls for Con A, which were incubated in Con A-ferritin in the presence of the inhibitory sugar (alpha-methyl-D-mannopyranoside), no ferritin particles were observed. The above-described procedure thus allows the localization of sugar residues in highly soluble pollen wall glycoproteins.     

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids

Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO4 after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO4. Morphometric analysis revealed that microwave enhanced fixation led to a larger mean cell volume, to larger cell surface area, and to larger surface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimicking exocytosis.     

Ruthenium red-mediated osmium binding for examining uncoated biological material under the scanning electron microscope

A simple technique for examining uncoated soft biological material under the scanning electron microscope is described. Rat tissues were initially fixed in 2.5% glutaraldehyde either by intravascular perfusion or by immersion. The samples were placed in buffered 2.5% glutaraldehyde containing 0.05% ruthenium red and postfixed in buffered 1% osmium tetroxide containing 0.05% ruthenium red. The samples were alternately incubated 3 to 5 times in 1% OsO4 and 0.1% ruthenium red solutions with continuous shaking at room temperature. The specimens were dehydrated, critical point dried, mounted and examined under the scanning electron microscope. Contour details were clearly defined at both the external and cut surfaces of the tissues. The specimens could be observed for more than 30 minutes without excessive charging or glow effects and the material remained stable under the beam at 20-25 kV and at various magnifications.     

Microtubule heterogeneity of Ornithogalum umbellatum ovary epidermal cells: non-stable cortical microtubules and stable lipotubuloid microtubules

Lipotubuloids, structures containing lipid bodies and microtubules, are described in ovary epidermal cells of Ornithogalum umbellatum. Microtubules of lipotubuloids can be fixed in electron microscope fixative containing only buffered OsO(4) or in glutaraldehyde with OsO(4) post-fixation, or in a mixture of OsO(4) and glutaraldehyde. None of these substances fixes cortical microtubules of ovary epidermis of this plant which is characterized by dynamic longitudinal growth. However, cortical microtubules can be fixed with cold methanol according immunocytological methods with the use of β-tubulin antibodies and fluorescein. The existence of cortical microtubules has also been evidenced by EM observations solely after the use of taxol, microtubule stabilizer, and fixation in a glutaraldehyde/OsO(4) mixture. These microtubules mostly lie transversely, sometimes obliquely, and rarely parallel to the cell axis. Staining, using Ruthenium Red and silver hexamine, has revealed that lipotubuloid microtubules surface is covered with polysaccharides. The presumption has been made that the presence of a polysaccharide layer enhances the stability of lipotubuloid microtubules.     

Tissue fixation and osmium black formation with nonvolatile octavalent osmium compounds.

Several compounds of osmiumVIII, including potassium osmiamate and coordination complexes of OsO4 with ammonia and various heterocyclic nitrogen compounds, have been synthesized and characterized. They have also been evaluated as substitutes for OsO4 in postfixation of biological specimens and in light and electron microscopic cytochemical methods resulting in osmium black formation. The most useful of these osmic compounds, a molecular addition complex of hexamethylenetetramine (methenamine) with OsO4, has a negligible vapor pressure of OsO4. It has the molecular formula C6H12N4.2OsO4 and has been designated osmeth. Although it has only limited solubility, aqueous solutions of the compound (or of OsO4) can be rapidly prepared by dissolution in a minimal amount of dimethylformamide and subsequent dilution with distilled water or buffer. Although stable in the solid state, the complex in solution undergoes partial dissociation releasing OsO4, and the odor of OsO4 becomes apparent. Such solutions of osmeth are (approximately 0.25%) considerably less concentrated with respect to OsO4 than solutions (1-2%) ordinarily employed for ultrastructural preservation or in cytochemical studies. Osmeth has limited value for postosmication after glutaraldehyde fixation because the generation (release) of OsO4 appears to be slow. Adequate osmication of tissue blocks exists only at the surface, but effective osmication can be achieved throughout tissue sections. In cytochemical reactions resulting in the formation of osmium blacks, the osmeth solutions are as effective as OsO4 solutions of equivalent concentrations. Our findings indicate that OsO4 solutions of less than 1% may be satisfactorily utilized in many cytochemical studies. Osmeth is safer and more convenient to handle than OsO4 because small amounts may be solubilized as needed. It should be considered as a substitute for OsO4 in ultrastructural cytochemistry. These results suggest that the effectiveness of OsO4 as a fixative may, in part, be related to its nonpolarity.The infrared spectra indicate that the OsO4 molecule is tetrahedral, perfectly symmetrical and, therefore, as a whole nonpolar. As a consequence, it could be expected to readily penetrate charged surfaces of tissues, cells, and organelles. The spectral studies show that osmeth is much less symmetrical and, to that extent, polar; thus, it penetrates biomembranes less readily.     

An ultrastructural study of complex carbohydrates in the posterior chamber and vitreous base of the mouse

Summary The fibrillar and mucoid extracellular matrix of the posterior chamber and vitreous base was studied in the mouse by electron microscopy using fixation and staining methods that demonstrated complex carbohydrates. These methods, including block-staining with Alcian Blue, allowed globular and filamentous hyaluronic acid, finely filamentous oligosaccharides, laminated glycolipids or lipophilic glycoproteins and stellate proteoglycan monomers to be identified tentatively. There was much less globular hyaluronic acid along the basement membrane of the peripheral retina and ciliary body than has been observed in the posterior fundus. A finely filamentous network on the basement membrane interconnected with a similar network covering individual collagen fibrils, zonules and meridional fibrillar laminae as well as with a branching fibrillar network that was seen in the posterior chamber and vitreous base. This interconnected system of fibrillar proteins and complex carbohydrates was also connected to the anterior hyaloid membrane. The infoldings of the ciliary epithelium contained stellage densities with characteristics of proteoglycan monomers similar to those reported in the matrix of cartilage. The complex carbohydrates of the posterior chamber and vitreous base are of several types known to affect protein function, provide water binding and assist in mechanical stability.     

The relationship between complex vesicles, dense-cored vesicles and dense projections in cortical synaptosomes

Cortical synaptosomes fixed in unbuffered OsO4 and glutaraldehyde have been block-stained with phosphotungstic acid (PTA) in order to investigate the relationship between complex vesicles and dense projections. It is concluded that the shell of the complex vesicles contributes to the formation of dense projections and that, in addition, there is a correspondence between this shell and the previously described presynaptic network. The process by which complex vesicles become associated with dense projections appears to be accentuated by electrical stimulation of the synaptosomes.     

Ruthenium Red Stain Able Surface Layer on Lung Alveolar Cells; Electron Microscopic Interpretation

Luft's ruthenium red (RR) method was applied to lung tissue. Small blocks of mouse lung were fixed for 1 hr with 1.2% glutaraldehyde at 0-4 C, buffered with 0.067 M cacodylate, pH 7.3 and containing RR, 1 mg/ml. Following fixation, lung blocks were immersed in 0.15 M cacodylate for 10 min and postfixed for 3 hr at room temperature with 2% OsO₄ buffered with 0.067 M cacodylate, pH 7.3, and containing RR, 1 mg/ml. Blocks were dehydrated with ethanol, embedded in Araldite, and ultrathin sections treated with uranyl acetate and lead citrate solutions to enhance contrast of cell structures. Electron micrographs revealed an electron-dense layer coating the exposed surfaces of alveolar cells. This layer corresponded in location and appearance to that observed by other investigators who used colloidal iron techniques.     

Parathyroid cell variants may be provoked during immersion fixation

Parathyroid glands of cattle, dogs, cats, mice and rats were immersed in glutaraldehyde or mixtures consisting of glutaraldehyde, formaldehyde and acrolein in either Na-phosphate, Na/K-phosphate or Na-cacodylate buffer, and postfixed with OsO4 in the same buffers or, alternatively, in s-collidine. Excellent preservation of bovine, feline and murine parathyroid glands was achieved with fixation mixtures containing 1% glutaraldehyde, 1.5-2% formaldehyde and 2.5-5% acrolein in 0.1 M Na-cacodylate with or without Ca2+ and Mg2+, Na-phosphate or Na/K-phosphate at 4 degrees C followed by postfixation with 1% OsO4 in the same buffers or in s-collidine containing sucrose, Ca2+ and Mg2+. This procedure largely abolished the occurrence of parathyroid cell variants. Bovine parathyroid glands were also satisfactorily preserved with 1% glutaraldehyde and 2% formaldehyde whereas 1% glutaraldehyde and 2.5 or 5% acrolein, lower or higher buffer osmolarity, or immersion at room temperature led to vacuolization of RER and to breakdown of membranes. In contrast, all fixation protocols led to the formation of dark and light cell variants and to multinucleated syncytial cells in dog and rat parathyroids. The results thus show that parathyroid cell variants arise during immersion fixation and that aldehydes, buffers and temperature are important factors for provoking parathyroid cell variants.     

Techniques to preserve soluble surface components in birch pollen wall: a scanning and transmission electron microscopic study

The exine of birch pollen was examined by scanning and transmission electron microscopy in the native state and after fixation in different aqueous fixatives: glutaraldehyde + OsO4; glutaraldehyde + cetylpyridinium chloride (CPC) + OsO4; glutaraldehyde + cuprolinic blue (CB); and periodate + lysine + paraformaldehyde (PLP). The native pollen exine showed a thin (3-5-nm) border of electron-dense material lining the tectum and electron-dense material within microchannels and bacula cavities. Fixation with the addition of CPC resulted in a voluminous surface coat surrounding the pollen grain, but empty microchannels and bacula cavities. After fixation with the addition of CB, there was a thin surface coat, whereas microchannels and bacula cavities were partially filled with electron-dense material. The other fixatives led to empty microchannels and bacula cavities. There was no surface coat on the pollen grain. However, after all fixation procedures, a thin electron-dense border of the tectum remained visible. Concerning the electron-dense material filling microchannels and bacula cavities in the native pollen grain, the results obtained in the present study suggest that it is either completely lost (after conventional and PLP fixation) or, after fixation with a precipitating additive, partially (CB) or completely (CPC) solubilized and precipitated on the surface of the pollen grain as a surface coat.     

Increase in the Calcium Content of Cardiac Tissue after Postfixation with Osmium Tetroxide

The concentration of osmium has been measured by destructive chemical analysis in glutaraldehyde fixed heart tissue postfixed with osmium tetroxide and embedded in epoxy resin. After such treatment, the mean atomic number of the specimen (Z) is close to 10, which permits a quantitative analysis of calcium (Ca) by the continuum method, using Z²/A as a correcting factor (A: atomic weight). Wavelength-dispersive X-ray microanalysis has been used to determine the Ca concentration of frog cardiac tissue fixed in glutaraldehyde and embedded in resin. These measurements have been repeated on tissue postfixed in osmium tetroxide; contrary to expectations, the apparent Ca concentration is much higher in osmium-treated than in nontreated tissue. However, this result is observed with OsO₄ solutions prepared in glass, not with solutions prepared in plastic. It is shown by energy dispersive X-ray analysis of droplets that OsO₄ solutions prepared in glass contain large amounts of calcium, potassium and silicon. Care must be taken in preparing OsO₄ fixatives when the fixed tissues are to be subjected to X-ray microanalysis of such elements as Ca or Si.     

The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

Ultrastructure of dyads in muscle fibers of Ascaris lumbricoides

The dyads of Ascaris body muscle cells consist of flattened intracellular cisternae applied to the sarcolemma at the cell surface and along the length of T-tubules. In specimens prepared by conventional methods (glutaraldehyde fixation, osmium tetroxide postfixation, double staining of sections with uranyl acetate and lead hydroxide), both the sarcolemma and the limiting membrane of the cisterna exhibit unit membrane structure and the space between them is occupied by a layer of peg-shaped densities which is referred to as the subsarcolemmal lamina. The lumen of the cisterna contains a serrated layer of dense material referred to as the intracisternal lamina. In specimens fixed in glutaraldehyde, dehydrated, and then postfixed in phosphotungstic acid, with no exposure to osmium tetroxide or heavy metal stains, the membranous components of the dyads appear only as negative images, but the subsarcolemmal and intracisternal laminae still appear dense. Except for the lack of density in membranes and in glycogen deposits, the picture produced by the latter method is very much like that of tissue prepared by conventional methods.