Morphogenesis in wing imaginal discs: its relationship to changes in the extracellular matrix

An extracellular matrix (ECM) lies between the upper and lower epithelial layers of the wing imaginal discs of moths. Organization and composition of this extracellular matrix, as revealed by staining with ruthenium red, tannic acid, and alcian blue, changes in concert with levels of hormones in the haemolymph. The ECM of the wing imaginal disc is an environment for cellular movements. Reorganization of the matrix and increase in ecdysteroid level is coupled with the proximal----distal migration of tracheal cells as well as the distal----proximal outgrowth of sensory neurons.     

Structural links between the renal stem/progenitor cell niche and the organ capsule

A special feature of the renal stem/progenitor cell niche is its always close neighborhood to the capsule during organ development. To explore this link, neonatal kidney was investigated by histochemistry and transmission electron microscopy. For adequate contrasting, fixation of specimens was performed by glutaraldehyde including tannic acid. The immunohistochemical data illustrate that renal stem/progenitor cells are not distributed randomly but are positioned specially to the capsule. Epithelial stem/progenitor cells are found to be enclosed by the basal lamina at a collecting duct (CD) ampulla tip. Only few layers of mesenchymal cells are detected between epithelial cells and the capsule. Most impressive, numerous microfibers reacting with soybean agglutinin, anti-collagen I and III originate from the basal lamina at a CD ampulla tip and line between mesenchymal stem/progenitor cells to the inner side of the capsule. This specific arrangement holds together both types of stem/progenitor cells in a cage and fastens the niche as a whole at the capsule. Electron microscopy further illustrates that the stem/progenitor cell niche is in contact with a tunnel system widely spreading between atypical smooth muscle cells at the inner side of the capsule. It seems probable that stem/progenitor cells are supplied here by interstitial fluid.     

Receptor-Mediated Calcium Influx in Chlamydomonas reinhardtii

ABSTRACT. Alcian blue acts as a secretagogue and chemorepellent in a variety of unicellular eukaryotes. We report that alcian blue stimulates flagellar excision and induction of RNA encoding flagellar proteins in Chlamydomonas reinhardtii . Flagellar excision by alcian blue is dependent on extracellular Ca²⁺ and is blocked by La³⁺, ruthenium red, and neomycin, and so is similar to flagellar excision by acid shock. However, the adf-l mutant excises its flagella following alcian blue treatment, but not following acid shock, thus genetically distinguishing alcian-blue-induced excision from acid-shock-induced excision. Wild-type, but not adf-1, cells regrow their flagella in the continued presence of alcian blue. Wild-type cells that regrow flagella in the presence of alcian blue fail to excise their flagella in response to either increased concentrations of alcian blue or to acid shock. Alcian blue treatment of cells also induces RNA encoding flagellar components, but in a manner distinct from other means of stimulation. These results suggest that treating Chlamydomonas with the secretagogue alcian blue initiates a Ca²⁺ influx pathway and that prolonged treatment with alcian blue desensitizes the acid-shock-activated Ca²⁺ influx pathway to acid treatment. Alcian blue will thus be a useful excitatory ligand in future studies of receptor-mediated Ca²⁺ signaling in the Chlamydomonas flagellar regeneration system.     

Histochemical staining of the complex carbohydrates of the midgut of the mosquito,Culex tarsalis coquillett

Histochemical staining of the midgut epithelial cell surface complex carbohydrates of the mosquito Culex tarsalis was examined electron microscopically. The microvillar surface is composed primarily of neutral vic-glycoconjugates; positively stained by silver methenamine and silver protein. Lanthanum and alcian blue staining indicate that the microvilli contain a minimal anionic component; possibly phosphoglycoconjugates. Similarly, the intercellular junctions contain a predominance of neutral vic-glycoconjugates. In addition, the intercellular junctions contain fixed positive charges, based on en bloc phosphotungstic acid staining. The midgut basolateral membrane system and the basal lamina are both highly anionic; stained by ruthenium red, tannic acid, alcian blue and periodic acid-chromic acid-phosphotungstic acid. The basolateral plasma membrane also contains some vic-glycoconjugates. Selective staining indicates that the anionic component of the basolateral plasma membrane and the basal lamina is predominantly carboxyl groups; no specific staining for sulfo- or phosphoglycoconjugates was observed.     

Chemically defined medium environment for the development of renal stem cells into tubules

The use of stem cells is a valuable therapeutical option for the regeneration of diseased tissues and organs. However, the involved cellular processes are hardly known. To gain detailed information about their development, a new culture technology was developed. Embryonic renal tissue containing stem/progenitor cells was mounted within a perfusion culture container at the interface of an artificial interstitium made of polyester. Using this innovative approach we show that renal tubules develop in chemically defined Iscove's modified Dulbecco's medium without serum addition and without coating by extracellular matrix proteins. The development of tubules depends on the administration of aldosterone, and can be visualized by immunohistochemical labeling. The presented technology makes the exact analysis of developmental steps now possible, and provides a new powerful tool to optimize growth and differentiation of renal stem cells. It may also enable many other kinds of stem cells to steer their development into functional tissues under clearly defined in vitro conditions.     

High-resolution visualization of the microbial glycocalyx with low-voltage scanning electron microscopy: dependence on cationic dyes.

The microbial glycocalyx is composed of a variety of polyanionic exopolysaccharides and plays important roles in microbial attachment to different substrata and to other cells. Here we report the successful use of low-voltage scanning electron microscopy (LVSEM) to visualize the glycocalyx in two microbial models (Klebsiella pneumoniae and Enterococcus faecalis biofilms) at high resolution, and also the dependence on fixation containing polycationic dyes for its visualization. Fixation in a paraformaldehyde-glutaraldehyde cocktail without cationic dyes was inadequate for visualizing the glycocalyx, whereas addition of various dyes (alcian blue, safranin, and ruthenium red) to the aldehyde cocktail appeared necessary for stabilization. The cationic dyes varied in size, shape, and charge density, and these factors appeared responsible for different phenotypic appearances of the glycocalyx with each dye. These results suggest that aldehyde fixation with cationic dyes for high-resolution LVSEM will be a useful tool for investigation of microbial biofilms as well as investigation of the extent and role of the glycocalyx in microbial attachment to surfaces.     

Tannic acid, Iron hematoxylin, Alcian blue, and Basic fuchsin for staining islets and reticular fibers of the pancreas

In this technique alpha cells are stained by basic fuchsin, beta cells by iron-hematoxylin, reticular fibers by ferric tannate, and much by alcian blue. Among 6 commonly used fixatives tested, Bouin's fluid fixation (8-12 hr) gave the best staining results. Procedure: paraffin sections to water; 0.5% Li₂CO₃ to remove picric acid; 20% tannic acid, 15 min; wash well; 2-4 sec in 0.5% basic fuchsin containing 10% alcohol; rinse, then differentiate in 1% aniline in 90% alcohol until alpha cells are red and beta cells pink; 1% phosphomolybdic acid, 1 min; 5% hematoxylin in 2% iron alum, 0.5 min; wash well; 1% filtered alcian blue SGX, 15 sec; rinse, dehydrate, clear, and mount in synthtic resin. Results: reticular fibers, black; acinar cells, orange to gray; alpha cells, red; collagenous fibers, red; beta cells, gray granules; ducts, bluish-green. The method was tested on rat, rabbit, dog, hamster, cow and man.     

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.     

The renal cortical fibroblast in renal tubulointerstitial fibrosis

Renal cortical fibroblasts have key roles in mediating intercellular communication with neighboring/infiltrating cells and extracellular matrix (ECM) and maintenance of renal tissue architecture. They express a variety of cytokines, chemokines, growth factors and cell adhesion molecules, playing an active role in paracrine and autocrine interactions and regulating both fibrogenesis and the interstitial inflammatory response. They additionally have an endocrine function in the production of epoetin. Tubulointerstitial fibrosis, the common pathological consequence of renal injury, is characterized by the accumulation of extracellular matrix largely due to excessive production in parallel with reduced degradation, and activated fibroblasts characterized by a myofibroblastic phenotype. Fibroblasts in the kidney may derive from resident fibroblasts, from the circulating fibroblast population or from haemopoetic progenitor or stromal cells derived from the bone marrow. Cells exhibiting a myofibroblastic phenotype may derive from these sources and from tubular cells undergoing epithelial to mesenchymal transformation in response to renal injury. The number of interstitial myofibroblasts correlates closely with tubulointerstitial fibrosis and progressive renal failure. Hence inhibiting myofibroblast formation may be an effective strategy in attenuating the development of renal failure in kidney disease of diverse etiology.     

The cell coat of the sensory and supporting cells of the rainbow trout saccular macula as demonstrated by reaction with ruthenium red and tannic acid.

The surface of most cells is covered by glycoconjugates. The composition and thickness of the surface coat varies among different cell types. The purpose of the present study was to demonstrate the presence of and to characterize the cell coat surrounding the cells in the saccular macula of the rainbow trout. Tissues were fixed in Karnovsky's fixative containing either ruthenium red (0.5, 1, or 2%) or tannic acid (1, 2, or 4%). The apical surface of the sensory and supporting cells reacted with both agents. Varying the concentration of the compounds within a certain range did not significantly affect the degree of tissue staining. Whereas ruthenium red staining was distributed evenly along the luminal surface of the epithelium and along the length of the stereocilia, tannic acid formed electron-dense clumps on the luminal surface of sensory and non-sensory cells and in the basal region of the macular epithelium. The stereocilia of the sensory cells also exhibited tannic acid-positive, electrondense precipitate, particularly near the distal ends of these processes, while uniform staining of the plasma membrane was seen along their lengths. The results of this study suggest that the trout saccular macula is provided with extracellular microenvironments which may be necessary for functional integrity.     

Cellular and molecular pathways that lead to progression and regression of renal fibrogenesis

Renal fibrosis is a common consequence and often a central feature of all the progressive renal diseases that lead to end-stage renal failure. In comparison to wound healing, during kidney fibrosis the length of the post-inflammatory phase often exceeds and continues unchecked resulting in scar formation. Infiltrating immune cells and a heterogeneous colony of interstitial cells derived from a variety of cellular origins such as resident mesenchymal cells, tubular epithelial cells, circulating fibrocytes, and bone marrow derived stem cells, communicate with each other and with inflamed and surviving parenchymal cells via a network of cytokines and adhesion molecules to populate the renal tubulointerstitial space during early fibrogenesis. Such fibroblasts subsequently secrete abundant extracellular matrix to achieve architectural remodeling in parallel with functional deterioration. Renal fibrosis is a dominant determinant of the clinical outcome of patients and yet for the most part, current therapies are ineffective or only marginally effective. This review highlights recent advances in our understanding of the cellular and molecular events leading to the progression of renal fibrosis.     

The epithelia of the protrusible tongue of Eurycea longicauda guttolineata (Hoolbrook 1838) (Urodela: Plethodontidae)

In this study the lingual and sublingual glands, the lingual stem and the epithelial surface of the protrusible secondary tongue were investigated by light, scanning and transmission electron microscopy. The quality of the secretions of the epithelia was characterized histochemically. The lingual epithelium is formed by superficial (pavement) and goblet cells and at the margin of the tongue pad are also regions covered by ciliated cells. On the dorsal part of the tongue there are goblet cells of type A with mainly acidic secretions and of type B containing neutral secretions. Most of the goblet cells on the ventral side of the tongue (hypoglottis) show a strong alcian blue/PAS positive reaction (type I) and some produce neutral secretions (type II). The glandular cells of the lingual gland react positively to alcian blue and PAS in the apical region of the gland. In contrast there is only alcian blue-positive staining in the basal part of the gland. The size and complexity of the inclusion bodies of the secretory granules increase in a basal direction. In addition, there are ciliated cells in the glandular epithelium. Although the epithelium of the lingual stem is thin, it is double-layered. The cell types observed in this region are identical to those of the ventral part of the protrusible tongue. At the margin of the sublingual gland are trough-like structures. In the center, tubular parts are observed. The cells of this gland are stain strongly with alcian blue (pH 1.0) mainly in the basal part of the gland. The results of this are compared to the tongue pad and the lingual gland of Salamandra salamandra and Ambystoma mexicanum.     

Dual staining of some sulfated mucopolysaccharides with alcian blue (pH 1.0) and ruthenium red (pH 2.5)

Summary A dual staining technique has been presented for the histochemical characterization of some sulfated mucopolysaccharides. It is a combined alcian blue (pH 1.0)-ruthenium red (pH 2.5) staining method which colors most sulfated mucopolysaccharides tested purple or purplish blue. A series of histochemical experiments using histological sections and casein films containing acidic polysaccharides of known chemical structure indicate that reactive sulfate and carboxyl groupings of polysaccharides are responsible, to an appreciable degree, for the alcianophilia and affinity towards ruthenium red of the substances respectively. A hypothesis is advanced as to the mechanism whereby ruthenium red binds anionic groupings of mucopolysaccharides.This investigation was supported by a Grant-in-Aid from the Japanese Ministry of Education (1968).     

Paracrine cellular and extracellular matrix interactions with mesenchymal progenitors during pulmonary alveolar septation

Alveolar development in humans primarily occurs postnatally and requires a carefully orchestrated expansion of distal epithelial and mesenchymal progenitor populations and coordinated differentiation, to create a highly segmented gas‐exchange surface. The regulation of alveolarization normally assimilates cues from paracrine cell–cell, cell–extracellular matrix, and mechanical interactions which are superimposed on cells and the extracellular matrix through phasic respiratory movement. In bronchopulmonary dysplasia, the entire process is precociously initiated when cellular and extracellular components are adapted to the saccular stage where movement and circulation are much more limited. This review focuses on mesenchymal cells (fibroblasts, endothelial cells, and pericytes), and epithelial cells are primarily discussed as sources of growth factor ligands or recipients of ligands produced by mesenchymal cells. Some interstitial fibroblasts differentiate to contractile myofibroblasts, containing a smooth muscle‐actin rich cytoskeleton, which connects with tensile and elastic elements in the extracellular matrix, and together comprise a load‐bearing network that diffuses mechanical forces during respiration. Other interstitial fibroblasts assimilate neutral lipid droplets, which regulate the differentiation of distal epithelial progenitors and surfactant production by alveolar type 2 cells. Pericytes organize and reinforce the capillary network as it expands to match the coverage of type 1 epithelial cells. Hyperoxia and the mechanical load imposed by positive pressure mechanical ventilation disrupt these paracrine interactions, leaving thickened alveolar walls, airways and arterioles, thereby diminishing gas‐exchange surface area. Better understanding of these mechanisms of alveolar septation will lead to more effective treatments to preserve and perhaps augment the surface usual sequence of events that drive alveolarization. Birth Defects Research (Part A) 100:227–239, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Role of TGF-β in chronic kidney disease: an integration of tubular,glomerular and vascular effects

Transforming growth factor beta (TGF-β) has been recognized as an important mediator in the genesis of chronic kidney diseases (CKD), which are characterized by the accumulation of extracellular matrix (ECM) components in the glomeruli (glomerular fibrosis, glomerulosclerosis) and the tubular interstitium (tubulointerstitial fibrosis). Glomerulosclerosis is a major cause of glomerular filtration rate reduction in CKD and all three major glomerular cell types (podocytes or visceral epithelial cells, mesangial cells and endothelial cells) participate in the fibrotic process. TGF-β induces (1) podocytopenia caused by podocyte apoptosis and detachment from the glomerular basement membrane; (2) mesangial expansion caused by mesangial cell hypertrophy, proliferation (and eventually apoptosis) and ECM synthesis; (3) endothelial to mesenchymal transition giving rise to glomerular myofibroblasts, a major source of ECM. TGF-β has been shown to mediate several key tubular pathological events during CKD progression, namely fibroblast proliferation, epithelial to mesenchymal transition, tubular and fibroblast ECM production and epithelial cell death leading to tubular cell deletion and interstitial fibrosis. In this review, we re-examine the mechanisms involved in glomerulosclerosis and tubulointerstitial fibrosis and the way that TGF-β participates in renal fibrosis, renal parenchyma degeneration and loss of function associated with CKD.     

Cationic dyes reveal proteoglycans on the surface of epithelial and endothelial kidney cells

The glomerular epithelial cells of the rat kidney fixed by vascular perfusion with an aldehyde solution containing either safranine O or alcian blue (and 0.3 M MgCl2) display filaments which are located close to the outer surface of the plasma membrane. These filaments are similar to those revealed by the same methods in the laminae rarae of the glomerular basement membrane. Alcian blue (and MgCl2) further demonstrates the presence of anionic sites inside the endothelial cell pores of the glomerular and peritubular capillaries, on the luminal surface of endothelial cells of large renal vessels and along the basolateral surface of the epithelial cells of the Bowman capsule and of the proximal convoluted tubule.     

The ultrastructure of secretion in the spermatheca of the salamander, Manculus quadrigitatus (Holbrook)

Spermathecae of mature salamanders (Manculus quadridigitatus) were studied by light and electron microscopy. Gross morphology exhibits a complex of muscle, connective tissue and pigment cells surrounding a cluster of tubules, which empty into a ciliated central duct leading to the cloacal cavity. The tubules are composed of myoepithetial cells and secretory cells, anchored to an encompassing basal lamina. Secretion products appear to be initiated as crystalline deposits, seen in mitochondrial repositories, which are subsequently sequestered in the perinuclear region. Observations show these vesicles to be precursors of the Golgi synthesis of carboxylated polysaccharides, as determined by histochemical tests using toluidine blue, ruthenium red, and alcian blue. The secretory products are emptied into the tubule lumen. thereby bathing the stored sperm and maintaining them in a viable state for approximately 8 months. The storage tubules appear to be a complete complex of varying epithelial cells specifically designed to support viable sperm and to resorb non-functional forms.     

Surface coats on human lymphocytes: freeze-drying and staining with cations.

Surface coats can be demonstrated on human peripheral blood lymphocytes by staining with ruthenium red, alcian blue, Thorotrast, and cationized ferritin, which are similar in distribution to a 40- to 65-nm layer of amorphous extracellular material recently reported on fixed, freeze-dried lymphocytes. Several additional lines of evidence, including X-ray microanalysis, suggest that the latter is not a contaminant added by freeze-drying. Freeze-drying may provide the means for a morphological assessment of the lymphocyte surface, including the extracellular coat, which may give additional insight into the immune response.