LANTHANUM STAINING OF THE SURFACE COAT OF CELLS : Its Enhancement by the Use of Fixatives Containing Alcian Blue or Cetylpyridinium Chloride

Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex.     

Ultrastructural and histochemical studies on the epithelium revestment layer in the tube feet of the starfish Asterina stellifera

The “cuticle,” which revests the starfish tube foot, has been studied by electron microscopy and the findings correlated with histochemical observations. The “cuticle” is composed by two distinct zones; an outer zone including numerous microvilli, which extend from the inner zone into and through a fibrillar substance distinctly organized in two layers. These microvilli protrude slightly beyond the outer surface, where their tips give rise tonumerous extremely delicate fibrils. The second inner zone, of quite variable thickness and condensation of material, presents a coarser fibrous matrix where organelles and inclusions can be found. The whole cuticular complex does not derive from the majority of the epithelial cells, but is probably an extension of a special kind of T-shaped cells appearing at intervals, the “cuticle” forming a syncytial surface. Histochemical investigations indicate that the “cuticle” contains a combination of neutral and acid mucopolysaccharide, with a marked neutral predominance, the outer one displaying also an extremely thin coat of acid mucopolysaccharide with the sulfate group. The ordered arrangement of the microvilli suggests that this situation is imposed by the strong bond existing between the microvilli and the ouble mucopolysaccharide layers which would act as a cementing substance stabilizing the entire apical surface of the cell.     

The surface coats on spores

There are surface coats on the sporocytes and spores of some pteridophytes and bryophytes which will bind one or more of the stains generally used to demonstrate the presence of an acid mucopolysaccharide surface coat in animal cells, viz. Alcian blue, colloidal iron, lanthanum, thorium, silver, SO,-coriphosphine and phosphotungstic acid. This suggests that the composition of the coat substances in the ferns and bryophytes agrees with that in animal cells. It has been found that thorium-staining in the sporocyte and the coat of the young spore of one fern, Botrychium lunaria , can be abolished with neuraminidase, indicating that sialic acid is a principal component of these coats. The spore wall in the pteridophytes (and probably also in the bryophytes) is constructed within these mucopolysaccharide surface coats, and it is suggested that differences in wall form are attributable to qualitative, quantitative and functional differences of the coats.     

Electron microscopic observations of the surface of L-cells in culture

Summary Techniques are described for the preparation of preshadowed replicas of both the upper and lower surfaces of L-cells in culture, and of cross sections of L-cells growing on a cellophane substrate. These revealed long slender microvilli, 800 to 1,100 A in diameter, projecting from both upper and lower surfaces of the cells. These microvilli were frequently observed to contact other cells and substrate, and to leave material behind on the substrate. The plasma membrane of the lower surface was separated from the substrate by an electron-lucent gap 200 to 300 A wide. The surface coat of the L-cell was visualized by staining with colloidal iron and ruthenium. Staining with colloidal iron was most intense on the surface of the microvilli. The gap between cell and substrate was intensely stained with ruthenium red. Enzymatic digestion of living cells revealed that both trypsin and neuraminidase reduced the staining of the cell coat by colloidal iron, whereas only trypsin altered its staining with ruthenium red. After trypsin treatment, fragments of an amorphous material with the staining characteristics of the cell coat were observed between the denuded cells. Treatment with ribonuclease, chymotrypsin or hyaluronidase did not affect the staining of the cell coat.     

Carbohydrate cytochemistry of the intestinal epithelium of Ascaris suum. Nature of the microvilli glycocalyx and basal lamella.

Results of various cytochemical tests demonstrate large deposits of glycogen within the intestinal absorptive cells of Ascaris suum. Carbohydrate material is also associated with the microvilli surface and basal lamella. Staining produced by the periodate-thiocarbohydrazide-osmium procedure was abolished by analine or m-aminophenol. Diastase digestion did not alter the staining on the microvilli surface. Similar results were seen using the silver methenamine procedure. A positive reaction was noted on the microvilli surface, vesicles in both the apical and basal cytoplasm, Golgi apparatus, and basal lamella. Lanthanum nitrate stained the microvilli surface and intercellular spaces between absorptive cells. Alcian blue or cetylpyridinium chloride in combination with lanthanum enhanced the staining produced by lanthanum alone. These results suggest the presence of acidic glycans on both the microvilli surface and basal lamella.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothiocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.     

Localization of glycoconjugates on the surfaces of pronephric tumor cells in vitro

Summary Differential localization of glycoconjugates was detected on microvilli and microridges of the intact cell surface of frog pronephric tumor cells in tissue culture. Alcian blue and Alcian blue/PAS staining showed a heavy concentration of dye limited to the unique short microvilli and extensive microridges of the tumor cells as previously seen with SEM (Tweedell and Williams 1976). Staining was absent or greatly reduced on microvilli of the normal pronephric cell surface. Previous exposure of each kind of cells to neuraminidase or extraction by mild hydrolysis removed the active staining sites but Alcian blue uptake was unaffected by prior digestion with testicular hyaluronidase. Fluorescein isothyocyanate (FITC) bound wheat germ agglutinin (WGA) produced a similar pattern of fluorescence on the microvilli of the tumor cells and a limited distribution on the normal cells. Digestion with neuraminidase preferentially removed but did not completely eliminate the surface binding of WGA on both the normal and tumor cells. Exposure of tumor cell monolayers to FITC bound limulin, a lectin specific for sialic acid, also produced an intense surface fluorescence on the microvilli and ridges of tumor cells. Prior treatment with neuraminidase prevented the surface fluorescence but not internal binding. Normal pronephric cells gave sparse surface fluorescence but extensive internal binding. Each procedure indicates a preferential localization of complex carbohydrates, including sialic acid, on the unique microvilli of the tumor cells. Concurrent assays for sialic acid recovered from the tumor cells indicated that lectin bound surface sialic acid was removable with neuraminidase.This study was supported by a grant from the Cancer Society of Saint Joseph County, Indiana and from the Phi Beta Psi Sorority     

Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

A COMPARATIVE STUDY OF THE ULTRASTRUCTURE OF MICROVILLI IN THE EPITHELIUM OF SMALL AND LARGE INTESTINE OF MICE

A comparative analysis of the fine structure of the microvilli on jejunal and colonic epithelial cells of the mouse intestine has been made. The microvilli in these two locations demonstrate a remarkably similar fine structure with respect to the thickness of the plasma membrane, the extent of the filament-free zone, and the characteristics of the microfilaments situated within the microvillous core. Some of the core microfilaments appear to continue across the plasma membrane limiting the tip of the microvillus. The main difference between the microvilli of small intestine and colon is in the extent and organization of the surface coat. In the small intestine, in addition to the commonly observed thin surface "fuzz," occasional areas of the jejunal villus show a more conspicuous surface coat covering the tips of the microvilli. Evidence has been put forward which indicates that the surface coat is an integral part of the epithelial cells. In contrast to the jejunal epithelium, the colonic epithelium is endowed with a thicker surface coat. Variations in the organization of the surface coat at different levels of the colonic crypts have also been noted. The functional significance of these variations in the surface coat is discussed.     

A fine structural analysis of the epidermis and cuticle of the oligochaete aeolosoma Bengalense stephenson

The fine structure of the epidermis and cuticle has been described for the oligochaete Aeolosoma bengalense. The epidermis is a pseudostratified epithelium and consists of the following cell types: ciliated and nonciliated supportive cells, pigment cells and associated satellite cells, mucous cells, basal cells, and ciliated non-supportive columnar cells. Overlying and restricted to the supportive cells is a delicate cuticle composed of: (a) a discontinuous layer of membrane-bounded surface particles; (b) a thin filamentous layer of moderate electron density just under the surface particles; (c) a thicker inner filamentous layer of low electron density. Digestion with pronase effectively removes the cuticle. This, together with the fact that it stains with alcian blue and ruthenium red, indicates that the cuticle contains an acid mucopolysaccharide. Regeneration of the cuticle, following pronase treatment, is marked by the elaboration of numerous microvilli by the supportive cells. Most of the microvilli are transitory and evidence supports a microvillar origin for the cuticular surface particles. The presence of cuticular surface particles may be a characteristic shared in common by all oligochaetes and, perhaps, some polychaetes.     

Electron microscope observations on the carbohydrate-rich cell coat present at the surface of cells in the rat

Periodic acid-silver methenamine, a fairly specific technique for glycoprotein detection, was used to stain a variety of rat tissues, in the hope of confirming the existence of a carbohydrate-rich "cell coat" at the surface of mammalian cells. It was found that nearly all cells are coated by a thin layer of stained material. Around fibrocytes and migrating blood cells, the layer is uniform and merges with the ground substance. In the nervous system, cells and processes are surrounded with a layer whose density increases in synaptic clefts. Around epithelial cells, the layer outlines apical microvilli, follows lateral interspaces, and extends between cells and basement membrane. The layer is continuous with the middle plate of desmosomes and can be followed within the wide portion of terminal bars. In contrast, staining usually vanishes when two adjacent plasma membranes fuse to form tight junctions. These findings indicate that the stained layer is a "cell coat" located outside the plasma membrane. Since the cell coat is also stained by colloidal thorium, a technique for detection of acidic carbohydrates, this structure presumably contains not only glycoprotein(s) but also acidic residues. The carbohydrates may play a role in holding cells together and in controlling the interactions between cells and environment.     

Identification and characterization of podocalyxin--the major sialoprotein of the renal glomerular epithelial cell

The glomerular epithelial polyanion is a specialized cell surface component found on renal glomerular epithelial cells (podocytes) that is rich in sialoprotein(s), as detected by staining with cationic dyes (colloidal iron, alcian blue) and wheat germ agglutinin (WGA). We have isolated rat glomeruli and analyzed their protein composition by SDS PAGE in 5-10% gradient gels. When the gels were stained with alcian blue or "Stains All," a single band with an apparent Mr of 140,000 was detected that also stained very prominently with silver, but not with Coomassie Blue. This band predominated in fluorograms of gels of isolated glomeruli that had been labeled in their sialic acid residues by periodate-[3H]borohydride. In lectin overlays, the 140-kilodalton (kd) band was virtually the only one that bound [125I]wheat germ agglutinin, and this binding could be prevented by predigestion with neuraminidase. [125I]Peanut lectin bound exclusively to the 140-kd band after neuraminidase treatment. An antibody was prepared that specifically recognizes only the 140-kd band by immunoprecipitation and immuneoverlay. By immunoperoxidase and immunogold techniques, it was localized to the surface coat of the glomerular epithelium and, less extensively, to that of endothelial cells. When analyzed (after electroelution from preparative SDS gels), the 140-kd band was found to contain approximately 20% hexose and approximately 4.5% sialic acid. These findings indicate that the 140-kd protein is the major sialoprotein of the glomerulus, and it is the only component of glomerular lysates with an affinity for cationic dyes and lectins identical to that defined histochemically for the epithelial polyanion in situ. Since this molecule is a major component of the cell coat or glycocalyx of the podocytes, we have called it "podocalyxin."     

Ultrastructural and histochemical differences in cell surface properties of strain-specific and nonstrain-specific TA3 adenocarcinoma cells

Transmission and scanning electron microscopy and histochemical and biochemical methods were used to investigate differences in cell structure and cell surface properties between the strain-specific TA3- St and nonstrain-specific TA3-Ha ascites sublines of the TA3 murine mammary adenocarcinoma. The TA3-St subline is lethal only to the syngeneic strain A mouse (the strain of origin), whereas the TA3-Ha subline is lethal even to foreign species. In contrast to the TA3-St cell surface, which has numerous folds and irregular microprojections, the TA3-Ha cell has abundant long microvilli of uniform dimensions. An extensive cell surface coat which resembles the "fuzz" coat found on microvilli of normal epithelium was present on the TA3-Ha, but not on the TA3-St cells. After routine fixation, the surface coat of the TA3- Ha cell usually appeared as a filamentous network extending 30-50 nm from the plasmalemma; occasionally, longer filamentous or rod-like structures were found extending 200-400 nm from the plasmalemma. The cell coat material was more extensive on the microvilli than on the intermicrovillous membranes. Free virus-like particles associated with TA3-Ha cells have a similar-appearing surface coat on their outer membranes. The density of surface anionic sites, determined with polycationic ferritin, was greater on the TA3-Ha than on the TA3-St cell surface, consistent with the presence at the TA3-Ha cell surface of several-fold more neuraminidase-susceptible sialic acid groups. The observed surface features of the nonstrain-specific TA3-Ha cell, in comparison to the strain-specific TA3-St cell, are consistent with the suggestion that sialic acid-rich glycoproteins at the TA3-Ha cell surface mask histocompatibility antigens and enhance the ability of malignant cells to invade foreign species.     

Histological and histochemical studies of the gall bladder of the graylag goose (Anser anser)

ABSTRACT

We investigated the histological structure of the graylag goose (Anser anser) gall bladder. Sections of the gall bladder were stained with hematoxylin and eosin (H & E), Alcian blue (pH 2.5) for acid mucopolysaccharides, Gomori’s method for reticular fibers, Masson’s trichrome, periodic acid-Schiff (PAS) and Verhoeff’s elastin stain. The goose gall bladder was composed of a tunica mucosa, tunica muscularis and tunica adventitia or tunica serosa. The tunica mucosa formed regularly distributed simple isometric folds plus larger, less numerous, branched folds. The luminal surface was lined by tall columnar epithelial cells that stained for both acid and neutral mucopolysaccharides. The epithelial cells formed a discontinuous striated border of interdigitating microvilli on the luminal surface. Neither a lamina muscularis nor goblet cells were observed in the tunica mucosa. Unusual findings included branched mucosal folds, discontinuous microvilli and absence of an outer longitudinal layer in the tunica muscularis. No marked sex-associated differences were found. The general histochemical and histological structures of the graylag goose gall bladder are similar to those of birds such as chukar partridge and quail, but with some unique elements that may reflect differences in organ function.     

Electron-microscopic study of the excretory system of hungry females of the tick Hyalomma asiaticum P. Sch. et E. Schl. 2]

In H. asiaticum the cells of the Malpighian tubules and these of the rectal cas have the uniform structure: the apical surface is covered with microvilli, the basal plasmatic membrane forms relatively small invaginations. As to ultrastructural characters, there is no distinct division of the Malpighian tubule into departments. The distal ends of the tubules are not only somewhat enlarged and form the so-called ampulla cells of which are noticeably flattened. The microvilli and basal folds of the plasmatic membrane in this area of the tubule are indistinct. The cells of the ampulla and the neighbouring area of the tubule are characterized by the presence of inclusions with mucopolysaccharide secretion confined by the membrane. The microvilli are most developed on cells of the proximal ends of the Malpighian tubules. Well developed microvilli of the rectal sac form a striated border each containing a microtube inside. The basal invaginations are developed here better than in the cells of the Malpighian tubules.     

Ultrastructural changes in the embryonic cells of the frog Microhyla ornata after cytochalasin H treatment

Biological effects of cytochalasin H (CH), a newly isolated mould metabolite, have been found to bring about disaggregation of embryonic cells and to inhibit cytokinesis. Disaggregation is known to be a phenomenon related to the cell surface. (The cells are held together by a mucopolysaccharide glycoprotein complex.) In the present work the fact that the mucopolysaccharide glycoprotein surface coat gets affected by CH treatment is confirmed by electron microscopy with the help of Lanthanum, a specific marker, which gets selectively absorbed to the cell surface material and renders it electron dense. The ultrastructural observations indicated the reduction of the cell surface material in treated embryos as compared to the controls. The reappearance of lanthanum-bound cell surface material in the recovered embryos was also observed. However, the exact mechanism of the action of CH on the cell surface remains to be clarified.     

A scanning electron microscopic study of larval development in the marine polychaete,Galeolaria caespitosa Lamarck (Serpulidae)

Summary Gametes and developing larvae of the polychaete Galeolaria caespitosa were examined by scanning electron microscopy. The sperm display a primitive morphology. When treated with 0.33 M CaCl₂, they release a branched acrosomal process. At spawning, the polygonal oocytes have a granular surface made up of spherules and the tips of microvilli. The oocyte coat develops a ridged appearance as the oocyte rounds up. At fertilization, the microvilli are withdrawn from the coat surface. Microvilli again appear on the coat surface during the trochophore stage, but the egg coat appears to be retained as the larval cuticle until the demersal stage. The surface of the larva now shows many microvilli. Details of the organization of several ciliary structures are clarified. Moreover, the present study shows rapid, sequential development of paired setal sacs, with the most anterior pair appearing first.     

Lycramine brilliant blue JL: a new stain for human megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte cytoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Lycramine Brilliant Blue Jl: A New Stain for Human Megakaryocytes

Using the acrylic textile dye Lycramine brilliant blue JL, mature and immature megakaryocytes from human bone marrow specimens stained metachromatically bright lavender. This coloration was not observed in other types of bone marrow cells. After digestion with either diastase or ribonuclease, subsequent staining of marrow specimens did not reveal a significant diminution of the intensity of staining of megakaryocytes. However, after incubation with hyaluronidase followed by staining with Lycramine brilliant blue JL, staining of megakaryocyte ctyoplasm was either imperceptible or very pale blue. Accordingly, at least one of the substances responsible for the staining reaction is acid mucopolysaccharide in the cytoplasm of megakaryocytes. With further experience and comparison with established immunologic and cytochemical techniques, staining of megakaryocytes with Lycramine brilliant blue JL may be a useful addition to the cytochemistry of blood and bone marrow cells.     

Ampullary organ morphology of freshwater salmontail catfish, Arius graeffei

Two types of ampullary organs are present in the skin of the freshwater salmontail catfish, Arius graeffei, each consisting of a short canal (0.2-0.5 mm) oriented perpendicular to the basement membrane and ending in an ampulla. Histochemical staining techniques (Alcian blue and Lillie's allochrome) indicate that the ampullary canals contain an acidic mucopolysaccharide gel, which is uniform in its staining properties along the canals. Type II ampullary organs consist of a canal, the wall of which is lined with cuboidal epithelial cells. The canal opens into an ampulla with 50-60 receptor cells. Electron microscopy reveals that the pear-shaped receptor cells bear microvilli on their luminal surface and lie adjacent to an unmyelinated neuron. Type III ampullary organs differ from Type II in that the canal wall consists of cells that possess a protein-rich sac at the luminal apex and have a polymorphic nucleus. The canals of Type III ampullary organs open to an ampulla with 8-30 receptor cells similar in both staining properties and structure to those of the Type II organ. In both types of ampullary organs, supportive cells surround each receptor cell except at the apex of the receptor cell.