Tannic Acid as an Electron Microscope Tracer for Permeable Cell Membranes

To recognize damaged cells in preparations for transmission electron microscopy, high molecular weight (1700 MW) tannic acid (1-4%) has been added to glutaraldehyde fixing solutions. During fixation, the tannic acid penetrates only those cells whose plasma membranes were previously damaged. It enhances the electron density of the injured cells, which become clearly distinguishable from the undamaged ones. As a tracer tannic acid shows great advantages over either lanthanum hydroxide, ruthenium red, or horseradish peroxidase. It diffuses evenly throughout the tissue block and is not removed by preparative steps. Furthermore, it is also a good tracer at the light microscope level.     

Cell junctions in the subcommissural organ of the rabbit as revealed by use of ruthenium red

Summary Tight junctions were found in the apical junctional complex of the adult rabbit subcommissural organ (SCO) in addition to zonulae adhaerentes and gap junctions of typical ependymal cells. Ventricular perfusion of ruthenium red before fixation was found to give excellent results for distinguishing between gap and tight junctions at the ependymal surface. The implication of tight junctions as a mechanical means of sealing off the SCO area from the cerebrospinal fluid and the use of ruthenium red as a tracer substance are discussed.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen.     

THE FINE STRUCTURE AND ELECTROPHYSIOLOGY OF HEART MUSCLE CELL INJURY

Injured frog heart cells electrically uncouple from their uninjured neighbors within 30 min after injury. This uncoupling process can be shown by the disappearance of an injury potential measured between such injured and uninjured cells. In the present study, the time course of the decline of injury potentials, and thus of electrical uncoupling, in bullfrog atrial trabeculae was determined. Tissue was fixed with glutaraldehyde and osmium tetroxide at various times after injury to determine the morphological changes which accompany this uncoupling process. In some cases, ruthenium red was included in the fixatives. Normal atrial cells are long and narrow, with intercellular junctions located along the lateral surfaces of the cells. Two types of intercellular junctions have been observed: cardiac adhesion plaques (CAPs), and close junctions. Close junctions occur only infrequently. Ruthenium red penetrates all around the cells, leaving only small areas within the CAPs unstained. After injury, the cells are very dense and the myofilaments disarranged. Both types of intercellular junction remain intact, and only slight changes within CAPs are observed. The results are discussed in relation to current concepts of intercellular communication.     

Peculiarities of the extracellular matrix in the interstitium of the renal stem/progenitor cell niche

The development of the nephron is piloted by interactions between epithelial and surrounding mesenchymal stem/progenitor cells. Data show that an astonishingly wide interstitial space separates both kinds of stem/progenitor cells. A simple contrasting procedure was applied to visualize features that keep renal epithelial and mesenchymal stem/progenitor cells in distance. The kidney of neonatal rabbits was fixed in solutions containing glutaraldehyde (GA) in combination with alcian blue, lanthanum, ruthenium red, or tannic acid. To obtain a comparable view to the renal stem/progenitor cell niche, the tissue was exactly orientated along the axis of collecting ducts. Fixation with GA or in combination with alcian blue or lanthanum revealed an inconspicuous interstitial space. In contrast, fixation with GA containing ruthenium red exhibits strands of extracellular matrix lining from epithelial stem/progenitor cells through the interstitium up to the surface of mesenchymal stem/progenitor cells. Fixation with GA containing tannic acid shows that the basal lamina of epithelial stem/progenitor cells, the adjacent interstitial space and also the surface of mesenchymal stem/progenitor cells are connected over a net of extracellular matrix. The applied technique appears to be a suitable method to illuminate the interstitium in stem/progenitor cell niches of specialized tissues, the microenvironment of tumors and extension of degeneration.     

Structure and permeability of goblet cell tight junctions in rat small intestine

Summary Two major cell types, goblet and absorptive cells, dominate the epithelial lining of small intestinal villi. We used freezefracture replicas of rat ileal mucosa to examine the possibility that tight junction structure, known to relate to transepithelial resistance, might vary with cell type. Tight junctions between absorptive cells were uniform in structure while those associated with villus goblet cells displayed structural variability. In 23% of villus goblet cell tight junctions the strand count was less than 4 and in 30% the depth was less than 200 nm. In contrast, only 4% of absorptive cell tight junctions had less than 4 strands and only 9% had depth measurements less than 200 nm. Other structural features commonly associated with villus goblet cell tight junctions but less commonly with absorptive cell tight junctions were: deficient strand cross-linking, free-ending abluminal strands, and highly fragmented strands. Bothin vivo ileal segments and everted loops were exposed to ionic lanthanum. Dense lanthanum precipitates in tight junctions and paracellular spaces were restricted to a subpopulation of villus goblet cells and were not found between villus absorptive cells. After exposure of prefixed ileal loops to lanthanum for 1 hour, faint precipitates of lanthanum were found in 14% of tight junctions and paracellular spaces between absorptive cells compared to 42% of tight junctions and paracellular spaces adjacent to villus goblet cells. When tested in Ussing chambers, the methods used for lanthanum exposure did not lower transepithelial resistance. Everted loops exposed to ionic barium and examined by light microscopy showed dense barium precipitates in the junctional zone and region of the paracellular space of villus goblet cells but not in these regions between absorptive cells. However, the macromolecular tracers, microperoxidase, cytochromec and horseradish peroxidase, were excluded from both villus goblet cell and absorptive cell paracellular spaces inin vivo segments. These findings suggest that a subpopulation of villus goblet cells may serve as focal sites of high ionic permeability and contribute to the relatively low resistance to ionic flow which characterizes the small intestinal epithelium.     

Localization of lanthanum tracer in oral epithelium using transmission electron microscopy and the electron microprobe

Although water soluble tracers have been used to localize intercellular permeability barriers with the transmission electron microscope, there is the possibility of translocation or loss of the tracer during processing. This study compares the localization of lanthanum tracer in keratinized oral epithelium after routine processing with lanthanum seen after using freeze drying to avoid aqueous fixation and dehydration. The electron probe was used to identify the lanthanum tracer in the tissue and to distinguish it from other electron-dense material. After incubating small biopsies of rat palate mucosa in 1% lanthanum nitrate, specimens were either routinely processed for electron microscopy or quick frozen, dehydrated, fixed in osmium vapour and infiltrated with epoxy resin. Examination in the transmission electron microscope indicated that preservation of the freeze dried tissue did not compare favourably with that of normally processed tissue, but the distribution of the electron-dense tracer in the intercellular spaces and the extent of the penetration through the epithelia was similar in the two types of preparations. Confirmation of the tracer as lanthanum was obtained by wavelength dispersive X-ray analysis with the electron probe. The results indicate that no appreciable shift in the localization of the tracer is introduced by routine aqueous fixation and dehydration for electron microscopic examination.     

ASSEMBLY OF GAP JUNCTIONS DURING AMPHIBIAN NEURULATION

Sequential thin-section, tracer (K-pyroantimonate, lanthanum, ruthenium red, and horseradish peroxidase), and freeze-fracture studies were conducted on embryos and larvae of Rana pipiens to determine the steps involved in gap junction assembly during neurulation. The zonulae occludentes, which join contiguous neuroepithelial cells, fragment into solitary domains as the neural groove deepens. These plaque-like contacts also become permeable to a variety of tracers at this juncture. Where the ridges of these domains intersect, numerous 85-Å participles apparently pile up against tight junctional remnants, creating arrays recognizable as gap junctions. With neural fold closure, the remaining tight junctional elements disappear and are replaced by macular gap junctions. Well below the junctional complex, gap junctions form independent of any visible, preexisting structure. Small, variegated clusters, containing 4–30 particles located in flat, particle-free regions, characterize this area. The number of particles within these arrays increases and they subsequently blend together into a polygonally packed aggregate resembling a gap junction. The assembly process in both apical and basal regions conforms with the concept of translational movement of particles within a fluid plasma membrane.     

Maturation of tight junctions in guinea-pig cecal epithelium

Summary By means of the freeze-fracture technique and in tracer studies it is demonstrated that the structure of tight junctions and the permeability to lanthanum of the guinea-pig cecal epithelium change during maturation of cells. Height and strand number of tight junctions in the apical-basal direction increase as crypt cells migrate to the surface of the epithelium. Likewise, the interlacing of continuous strands was greater in surface than in crypt junctions. The numerous free-ends, isolated individual freestrands and maculae occludentes found in crypt cells were absent in surface epithelial cells. Goblet cells, located at the bottom of crypts, displayed tight junctions similar in characteristics to those of cells located in the middle region of crypts. Cells at the surface and in middle regions of crypts possess tight junctions impermeable to lanthanum, whereas junctions between cells located at the bottom of crypts often were permeable to the tracer, indicating that permeability decreases as the epithelial cells mature. Genesis and maturation mechanisms related to structural configuration of tight junctions are discussed.     

A novel occluding junction which lacks membrane fusion in insect testis

Spermatocysts develop within the lumina of the lepidopteran testis. Each spermatocyst contains a clone of maturing germ cells which are separated from the fluid in the testicular lumen by a layer of somatic envelope cells. A blood-testis barrier is located at the level of the somatic envelope cells. We used macromolecular tracers horseradish peroxidase (applied before fixation) and ruthenium red (applied during fixation) with thin sections and freeze-fracture replicas to study the nature of this barrier in spermatocysts of the tobacco budworm, Heliothis virescens. Movement of the tracers into the spermatocysts was blocked by a structure at the outer edge of the septate junctions which join the spermatocyst envelope cells. In freeze-fracture replicas there was a P-face ridge or an E-face groove in this location. The ridge/groove appeared similar to a single-stranded vertebrate tight junction. Unlike tight junctions, however, there was no fusion or even close apposition of adjacent cell membranes in this location. We conclude, therefore, that a novel type of occluding junction was the barrier to paracellular movement of macromolecules in Heliothis spermatocysts.     

The penetration of exogenous tracers through the enameloid organ of developing teleost fish teeth

In order to determine whether exogenous materials permeate to the forming tooth enameloid matrix, teleost species were injected intramuscularly with horseradish peroxidase (HRP) or myoglobin, or; intracardially with lanthanum nitrate or HRP, then killed a predetermined intervals post-injection. Tooth bearing bones were processed for transmission electron microscopy. At the enameloid matrix formation stage, capillaries associated with the enameloid organ were few in number and rarely fenestrated. Both organic tracers reached the matrix at cervical but not coronal, regions of the teeth in all species examined. Lanthanum was rarely observed extravascularly and never extended to the enameloid matrix at the secretion stage. At the enameloid mineralization stage, fenestrated capillaries were closely associated with the outer dental epithelial cells (ODE). All tracers were observed in the plasma membrane invaginations of the ODE. Only intracardially injected HRP compromised the apical intercellular junctions of the inner dental epithelial cells (IDE) to reach the mineralizing enameloid Lanthanum did not extend past the ODE-IDE cell junctions. It is concluded that the close association of mineralization stage fenestrated capillaries with the highly invaginated ODE cells result in increased tracer penetration compared to the secretory stage. The deeper penetration of the organic tracers, compared with lanthanum, between mineralization stage IDE cells may be due to longer in vivo circulation of the former material. The apical junctions of mineralization stage IDE cells, however, remained impermeable to the organic tracers. The absence of mineral in secretory stage enameloid mineral could not be due to specialized cell junctions preventing access of molecules to the matrix. It is suggested that controlling factors other than cellular permeability initiate enameloid mineralization.     

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% O₃O₄ 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.     

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.     

The fine structure of the glycocalyx of equine spermatozoa: a high-resolution cytochemical study.

Equine spermatozoa were obtained from ejaculates of young stallions. The seminal plasma was removed and the sperm pellets washed three times with 0-15 M-NaCl solution before final centrifugation at 4500 g for 15 min. The pellets were fixed in a mixture of 2-5% glutaraldehyde in 0-1 M-cacodylate buffer, pH 7-4, with 0-5% Alcian blue and post-fixed in 1% osmium tetroxide with 1% lanthanum nitrate; other samples were treated with ruthenium red. All samples were dehydrated in ascending concentrations of ethanol, embedded in araldite and thin sections examined in an electron microscope. Electron dense deposits of lanthanum were present on the surface plasmalemma of the head, mid-piece and tail of 70% of mature spermatozoa, and similar deposits were seen in ruthenium red-treated samples. No glycocalyx was observed in untreated spermatozoa.     

Studies on transmembrane and paracellular phenomena in the foot of the slug Agriolimax reticulatus (Mü)

Summary Using the enzyme peroxidase and ionic lanthanum as tracers, paracellular uptake has been demonstrated in the foot of the slug Agriolimax reticulatus (Mü). Both tracers appeared to pass between adjacent foot epithelial cells and were demonstrated in the zonula adhaerens, the septate desmosomes, and the intercellular spaces which occur beneath the septate junctions. Ferritin, a somewhat larger tracer, was excluded from all these sites.Ionic lanthanum was not normally pinocytosed in short incubation times. The epithelial cells could be induced to endocytose this marker, however, when combined with a variety of proteins. The implications these findings have on the uptake of molluscicides is discussed.This research was supported by the Agricultural Research Council (G.B.) Grant No. AG 72/13     

Ultrastructural study of tracer permeability through the cat and ferret enamel organ

The access of exogenous materials to the developing enamel surface has been intensively studied in rodents, but not in other mammalian species. This ultrastructural study investigates the permeability of injected horseradish peroxidase (HRP) and lanthanum tracers in cat and ferret tooth buds. In cat enamel organs fixed by immersion, lanthanum did not escape the capillaries overlying secretory stage tooth buds, but it did permeate up to the distal junctions of ruffle-ended (RA) and the proximal junctions of smooth-ended (SA) ameloblasts. Perfusion fixation with lanthanum compromised junctional integrity of cat ameloblasts at all stages of development. Similarly, HRP rarely escaped the capillaries associated with cat secretory stage enamel organs. However, unlike lanthanum, HRP was mostly confined to the vasculature of maturation stage enamel organs in immersion fixed cats at all time intervals examined. In ferrets, HRP penetrated up to, but not beyond, the distal junctional complexes of secretory ameloblasts. In maturation stage enamel organs, HRP coated the papillary and RA cells, but did not penetrate the RA distal cell junctions. HRP did permeate the extracellular spaces of SA to reach the underlying enamel surface. Ameloblasts in transitional phases of SA and RA endocytosed HRP at the distal cell surface. This data leads to several conclusions. First, HRP localization in the ferret paralleled that observed in rodents. Second, the results of cat enamel organs substantiate previous studies showing perfusion fixation can increase vascular and intercellular permeability to lanthanum. However, in cats fixed by immersion, both lanthanum and HRP were restricted to capillaries associated with the secretory stage enamel organ, and only lanthanum escaped maturation stage capillaries. It is suggested that variations in the fenestrations and distribution of capillaries associated with the cat enamel organ may differentially retain some materials and permit other materials to escape with relative ease.     

Cardiac gap junction configuration after an uncoupling treatment as a function of time

Rabbit ventricle either was fixed in glutaraldehyde without injury (control) or was injured before fixation, presumably causing electrical uncoupling of the gap junctions. All tissue was then processed for freeze-fracture. Replicas of control gap junctions exhibited irregular packing of the P-face particles and E-face pits. Average center-to-center spacing of the particles was 10.5 nm. Tissue fixed 1-5 min after injury showed clumping of gap junctional particles and pits. Within the clumps, the particles and pits were hexagonally packed and the center-to-center spacing of the particles averaged 9.5 nm. In tissue fixed 15-30 min after injury, the clumps of gap junctional particles had coalesced into a homogeneous structure in most junctions. The packing of the particles and pits was hexagonal and the spacing of the particles averaged 9.5 nm. A few pieces of rabbit atrium were frozen without prior fixation or cryoprotection to try to assess the effect of glutarldehyde fixation on gap junction structure. In this tissue the gap junctional particles were irregularly packed and their spacing averaged 10.0 nm.     

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.     

The gastric mucosal barrier: structure of intercellular junctions in the dog

The canine gastric mucosa consists of two regions, the surface mucous cells and gland area cells including parietal, chief, and mucous-containing cells. We have used quantitative freeze-fracture methods in conjunction with thin-section extracellular tracers to document and correlate tight junction morphology with epithelial permeability. The number of strands in the tight junction complexes of the surface cells and gland cells is the same, but differences in strand arrangement exist. The surface cells have an interwoven tight junction configuration which is impermeable to extracellular tracers. The gland cell junctions are regularly arranged and often permeable to extracellular lanthanum. The possibility that the observed difference in permeability between the tight junctions of the surface mucous cells and those of the gland cells is related to their structural configuration is discussed.     

The demonstration of close nerve-Purkinje fibre contacts in false tendons of sheep heart

Summary An observation of intimate nerve-Purkinje fibre associations in false tendons of sheep heart is reported. Nerve bundles were observed in deep clefts of Purkinje fibres, in channels running between coupled Purkinje cells and embedded within Purkinje cells, as well as in the outer connective tissue sheath. Most nerve terminals in these areas were filled with small clear vesicles and a few large dense-cored vesicles. Only a few axons with many small dense-cored vesicles were observed.Intimate associations (separation, 60 to 90 nm) between the Purkinje cell and nerve varicosity were observed in the deep clefts. Similar close appositions were also present where nerves were embedded in Purkinje cells. In these cases the Purkinje cell enclosing the nerve bundle formed intercellular junctions with its own sarcolemma.Elaborate sarcolemmal folds with multi-vesicular bodies were also frequently observed near nerve bundles and varicosities. The identity of the transmitter is unknown although the nerves forming intimate associations with Purkinje cells have a morphology typical of cholinergic nerves.     

Ultrastructure and permeability of lymph node microvasculature in the mouse

Summary The microvasculature of lymph nodes and Peyer's patches consists of arterioles, capillaries and venules. The postcapillary segment comprises high-endothelial venules (HE venules) as well as ordinary venules. In order to study the ultrastructure of the microvasculature, particularly with respect to the nature of intercellular junctions, lanthanum and ruthenium red were used as tracers. Furthermore, to evaluate the permeability properties of the different segments of the microvasculature, intravenously injected horseradish peroxidase (HRP; MW: 40,000) was used.All segments of the microvasculature are permeable to HRP. However, the mechanism of transport across the vascular wall varies in the different segments, apparently correlated with a gradual decrease in number of transport vesicles and a gradual attenuation in the sealing of the endothelial cells. Tight junctions are present in arterioles, and it is assumed that HRP reach the basal lamina exclusively by vesicular transport. Incomplete or focal tight junctions are present in the capillaries, and both intercellular and vesicular pathways are observed. In the venules the intercellular pathway seems to be the dominant one, while vesicular transfer is negligible. However, some micropinocytic vesicles in the HE venule endothelial cells probably represent the initial stage of an intracellular digestion.