Aqueous solutions of lead salts (1, 2) and saturated solutions of lead hydroxide (1) have been used as stains to enhance the electron-scattering properties of components of biological materials examined in the electron microscope. Saturated solutions of lead hydroxide (1), while staining more intensely than either lead acetate or monobasic lead acetate (l , 2), form insoluble lead carbonate upon exposure to air. The avoidance of such precipitates which contaminate surfaces of sections during staining has been the stimulus for the development of elaborate procedures for exclusion of air or carbon dioxide (3, 4). Several modifications of Watson's lead hydroxide stain (1) have recently appeared (5-7). All utilize relatively high pH (approximately 12) and one contains small amounts of tartrate (6), a relatively weak complexing agent (8), in addition to lead. These modified lead stains are less liable to contaminate the surface of the section with precipitated stain products. The stain reported here differs from previous alkaline lead stains in that the chelating agent, citrate, is in sufficient excess to sequester all lead present. Lead citrate, soluble in high concentrations in basic solutions, is a chelate compound with an apparent association constant (log Ka) between ligand and lead ion of 6.5 (9). Tissue binding sites, presumably organophosphates, and other anionic species present in biological components following fixation, dehydration, and plastic embedding apparently have a greater affinity for this cation than lead citrate inasmuch as cellular and extracellular structures in the section sequester lead from the staining solution. Alkaline lead citrate solutions are less likely to contaminate sections, as no precipitates form when droplets of fresh staining solution are exposed to air for periods of up to 30 minutes. The resultant staining of the sections is of high intensity in sections of Aralditeor Epon-embedded material. Cytoplasmic membranes, ribosomes, glycogen, and nuclear material are stained (Figs. 1 to 3). STAIN SOLUTION: Lead citrate is prepared by

/pdf/the-use-of-lead-citrate-at-high-ph-as-an-electron-opaque-260nso1ftx.pdf

THE USE OF LEAD CITRATE AT HIGH pH AS AN ELECTRON-OPAQUE STAIN IN ELECTRON MICROSCOPY

Epoxy embedding methods of Glauert and Kushida have been modified so as to yield rapid, reproducible, and convenient embedding methods for electron microscopy. The sections are robust and tissue damage is less than with methacrylate embedding.

/pdf/improvements-in-epoxy-resin-embedding-methods-17w13zwqe8.pdf

Improvements in epoxy resin embedding methods.

The aldehydes introduced in this paper and the more appropriate concentrations for their general use as fixatives are: 4 to 6.5 per cent glutaraldehyde, 4 per cent glyoxal, 12.5 per cent hydroxyadipaldehyde, 10 per cent crotonaldehyde, 5 per cent pyruvic aldehyde, 10 per cent acetaldehyde, and 5 per cent methacrolein. These were prepared as cacodylate- or phosphate-buffered solutions (0.1 to 0.2 M, pH 6.5 to 7.6) that, with the exception of glutaraldehyde, contained sucrose (0.22 to 0.55 M). After fixation of from 0.5 hour to 24 hours, the blocks were stored in cold (4°C) buffer (0.1 M) plus sucrose (0.22 M). This material was used for enzyme histochemistry, for electron microscopy (both with and without a second fixation with 1 or 2 per cent osmium tetroxide) after Epon embedding, and for the combination of the two techniques. After fixation in aldehyde, membranous differentiations of the cell were not apparent and the nuclear structure differed from that commonly observed with osmium tetroxide. A postfixation in osmium tetroxide, even after long periods of storage, developed an image that—notable in the case of glutaraldehyde—was largely indistinguishable from that of tissues fixed under optimal conditions with osmium tetroxide alone. Aliesterase, acetylcholinesterase, alkaline phosphatase, acid phosphatase, 5-nucleotidase, adenosine triphosphatase, and DPNH and TPNH diaphorase activities were demonstrable histochemically after most of the fixatives. Cytochrome oxidase, succinic dehydrogenase, and glucose-6-phosphatase were retained after hydroxyaldipaldehyde and, to a lesser extent, after glyoxal fixation. The final product of the activity of several of the above-mentioned enzymes was localized in relation to the fine structure. For this purpose the double fixation procedure was used, selecting in each case the appropriate aldehyde.

/pdf/cytochemistry-and-electron-microscopy-the-preservation-of-kpmoxmynkl.pdf

Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation.

The epithelia of a number of glands and cavitary organs of the rat and guinea pig have been surveyed, and in all cases investigated, a characteristic tripartite junctional complex has been found between adjacent cells. Although the complex differs in precise arrangement from one organ to another, it has been regularly encountered in the mucosal epithelia of the stomach, intestine, gall bladder, uterus, and oviduct; in the glandular epithelia of the liver, pancreas, parotid, stomach, and thyroid; in the epithelia of pancreatic, hepatic, and salivary ducts; and finally, between the epithelial cells of the nephron (proximal and distal convolution, collecting ducts). The elements of the complex, identified as zonula occludens (tight junction), zonula adhaerens (intermediary junction), and macula adhaerens (desmosome), occupy a juxtaluminal position and succeed each other in the order given in an apical-basal direction. The zonula occludens (tight junction) is characterized by fusion of the adjacent cell membranes resulting in obliteration of the intercellular space over variable distances. Within the obliterated zone, the dense outer leaflets of the adjoining cell membranes converge to form a single intermediate line. A diffuse band of dense cytoplasmic material is often associated with this junction, but its development varies from one epithelium to another. The zonula adhaerens (intermediate junction) is characterized by the presence of an intercellular space ( approximately 200 A) occupied by homogeneous, apparently amorphous material of low density; by strict parallelism of the adjoining cell membranes over distances of 0.2 to 0.5 micro; and by conspicuous bands of dense material located in the subjacent cytoplasmic matrix. The desmosome or macula adhaerens is also characterized by the presence of an intercellular space ( approximately 240 A) which, in this case, contains a central disc of dense material; by discrete cytoplasmic plaques disposed parallel to the inner leaflet of each cell membrane; and by the presence of bundles of cytoplasmic fibrils converging on the plaques. The zonula occludens appears to form a continuous belt-like attachment, whereas the desmosome is a discontinuous, button-like structure. The zomula adhaerens is continuous in most epithelia but discontinuous in some. Observations made during experimental hemoglobinuria in rats showed that the hemoglobin, which undergoes enough concentration in the nephron lumina to act as an electron-opaque mass tracer, does not penetrate the intercellular spaces beyond the zonula occludens. Similar observations were made in pancreatic acini and ducts where discharged zymogen served as a mass tracer. Hence the tight junction is impervious to concentrated protein solutions and appears to function as a diffusion barrier or "seal." The desmosome and probably also the zonula adhaerens may represent intercellular attachment devices.

/pdf/junctional-complexes-in-various-epithelia-9u1kkdzfat.pdf

Junctional complexes in various epithelia

In the course of an electron microscopic study of the peripheral region of the skeletal muscle fiber of the frog, the presence of certain cells, intimately associated with the muscle fiber, have been observed which we have chosen to call satellite cells. Since these cells have not been reported previously and indeed might be of interest to students of muscle histology and furthermore, as we shall suggest, might be pertinent to the vexing problem of skeletal muscle regeneration, a brief communication describing this finding is warranted prior to a more detailed study. The observations reported here have been made on bundles of fibers dissected from the tibialis anticus muscle of the frog. The material has been fixed by the conventional method with osmium tetroxide, and the embedding has been carried out with methacrylate and with epoxy (epon) resin. In sections that were \"stained,\" the lead hydroxide solution of Watson (1) was used. As seen in the attached electron micrograph of the satellite cell, the striking paucity of cytoplasm relative to its nucleus results in the cell assuming the shape of the nucleus. In fact, it is virtually impossible to discern the cellular nature of this entity in the light microscope, as it appears to be indistinguishable from a peripheral muscle nucleus proper. In electron micrographs the cell is seen \"wedged\" between the plasma membrane of the muscle fiber and the basement membrane, which invests the fiber throughout its length in close association with the plasma membrane. The intimacy of this satellite cell with respect to the multinucleate muscle cell is further revealed in the fact that, in general, the surface of the muscle fiber is not distorted outward but instead the satellite cell protrudes inward pushing the myofibrils of the muscle cell aside. On the inner surface, the plasma membrane of the satellite cell is in appositon with the plasma membrane of the muscle cell. Unfortunately, because of the limited observations and the difficulty in acquiring sufficient data readily with electron micrographic techniques, it is not possible at present to estimate the frequency of occurrence of these cells in a typical muscle fiber in our preparation of tibialis anticus muscle. The only generalization warranted at this time is that the peripheral muscle nuclei proper occur much more frequently than the satellite cells. It is interesting that upon alerting other investigators to these findings, similar cells have been found in electron micrographs of two other muscles of the frog, namely sartorius (2) and ileofibularis (3), and of the sartorius and tongue muscle of the white rat (4). (Though the direct evidence is restricted to these two vertebrates, it seems reasonable to hazard a guess that skeletal muscle fibers of vertebrates in general contain satellite cells.) It is tempting to speculate about the origin and the role of the satellite cells. Before stating the several possible hypotheses that have figured in our interpretations, it is pertinent to recall a most striking characteristic of regenerating muscle fibers in the least ambiguous case where the sarcolemma-tube remains intact, the myoplasm having undergone hyaline formation and retraction as a result of trauma. Within 48 hours a marked presence of \"free cells\" is noted in the empty tube, the cells appearing both as \"round\" and \" f u s i f o r m \" types (5). Moreover, in tissue culture studies of mature skeletal muscle explants, fi'ee cells are also seen emanating from the explant. The central question must be asked: what is the origin of these cells? Most cytologists lean toward the interpretation that surviving nuclei in the damaged multinucleate muscle cell give rise to single cells by \"gathering up\" cytoplasm from the sarcoplasm of the muscle cell--an unusual mechanism, however, for vertebrate systems. If this point of view is taken, the first and immediate hypothesis suggests itself, namely, that in the resting state some cells are being produced at a slow rate by the above mechanism and reside just outside the plasma membrane of the muscle cell, and that upon being stimulated by trauma, e.g. ischemia, mechanical compression, toxic agents, etc., the rate of production of such cells is increased. The second hypothesis, more in keeping with conventional notions of cytology, is that the satellite cells are remnants from the embryonic development of the multinucleate muscle cell which results from the process of fusion of individual myoblasts. Thus the satellite cells are

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Satellite cell of skeletal muscle fibers.

Heavy metals may be incorporated from solution into tissue sections for electron microscopy The resulting increase in density of the tissue provides greatly enhanced contrast with minimal distortion Relative densities of various structures are found to depend on the heavy metal ions present and on the conditions of staining Certain hitherto unobserved details are revealed and some sort of specificity exists, although the factors involved are not yet understood

https://rupress.org/jcb/article-pdf/4/4/475/1069933/475.pdf

Staining of Tissue Sections for Electron Microscopy with Heavy Metals

Descriptions of three heavy metal stains and methods of application to tissue sections for electron microscopy are presented. Lead hydroxide stains rather selectively two types of particles in liver: those associated with the endoplasmic reticulum and containing ribonucleic acid and other somewhat larger particles. Barium hydroxide emphasizes certain bodies within vesicles of the Golgi region of hepatic cells. Alkalized lead acetate is useful as a general stain, as are also lead and barium hydroxides.

https://rupress.org/jcb/article-pdf/4/6/727/1385577/727.pdf

Staining of Tissue Sections for Electron Microscopy with Heavy Metals II. Application of Solutions Containing Lead and Barium

The term pore complex is proposed for approximately cylindrical formations which are observed with the electron microscope to penetrate the nuclear envelope of cells. Cross-sections of the pore complex are somewhat annular in shape, but differ in appearance depending upon the level of the cross-section with respect to the nuclear surface. An explanation is offered for the apparent discrepancy between the width of pores in sections perpendicular to the nuclear envelope and the width of cross-sections of the pore complex in tangential sections. Channels associated with the pore complex extend deep into the nucleus. Although crescents and spirals of ribonucleoprotein particles were often seen in the immediate vicinity of the outer nuclear membrane, direct association with the pore complex was not observed. Many examples were found of pores that were not covered by a continuous membrane although the possibility of such a covering in some cases is not precluded.

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Further Observations on the Nuclear Envelope of the Animal Cell

/pdf/observations-on-a-granule-associated-with-chromatin-in-the-1hrgbnttql.pdf

Michael L. Watson

Papers

Staining of Tissue Sections for Electron Microscopy with Heavy Metals

Staining of Tissue Sections for Electron Microscopy with Heavy Metals II. Application of Solutions Containing Lead and Barium

Further Observations on the Nuclear Envelope of the Animal Cell

Observations on a granule associated with chromatin in the nuclei of cells of rat and mouse.

The development of the hamster lower incisor as observed by electron microscopy.