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).     

Positive periodic acid-Schiff staining of acid mucopolysaccharides

Synopsis Evidence is presented to demonstrate that acid mucopolysaccharides will stain with the periodic acid-Schiff technique following prolonged oxidation with periodic acid. Smears of purified acid mucopolysaccharides begin to stain slightly with the Schiff reagent after 4 hr of periodate oxidation and reach an optimal staining intensity some time between 7 and 16 hr. The acid mucopolysaccharides in umbilical cord and cock's comb sections begin to stain at about 7 hr, reaching an optimum between 16 and 24 hr. It is suggested that the mechanism of staining of acid mucopolysaccharides in the PAS technique appears to derive from cleavage oftrans groups of the hexuronic acid fraction which require prolonged oxidation since the theoretical more yield is reached with difficulty. Moreover, it would appear that the mechanism of staining with glycogen under the usual conditions of PAS staining perhaps derives from end-group oxidation and subsequent Schiff staining of the engendered aldehydes since glycogen is almost entirely in thetrans configuration. Acid mucopolysaccharides, on the other hand, will not stain uncer the usual conditions as they appear to have a negligible proportion of end groups.     

Sulfated acid mucopolysaccharides in the cortical granules of eggs. Effects of quaternary ammonium salts on fertilization

Sulfated acid mucopolysaccharides have been shown to be constituents of cortical granules in sea urchin and vertebrate eggs. These observations were made possible by retaining soluble acid mucopolysaccharides in situ within the eggs by precipitation during fixation with cetyltrimethyl-ammonium bromide, a quaternary ammonium salt. The sulfated mucopolysaccharides were then identified by staining with Astrablau at pH 0.2 and also by reaction with sodium rhodizonate. Staining reaction with Alcian blue at pH 2.5 showed that carboxylated mucopolysaccharides may also be present in cortical granules. The natural ionic environment of these eggs would favor the formation of very stable complexes between sulfated mucopolysaccharides and quaternary ammonium salts. Brief exposure of unfertilized sea urchin eggs to several quaternary ammonium compounds produced a residual adverse effect on subsequent fertilization in terms of increased vulnerability to polyspermy and reduced fertilizability. These results suggest that sulfated acid mucopolysaccharides participate in the function of the cortical granules and the establishment of the block to polyspermy at fertilization, and possibly in other cellular secretory processes.     

A study of acid mucopolysaccharides in cold microtome sections of normal and experimentally hydrated bovine corneas

Summary Acid mucopolysaccharides were investigated in cold microtome sections of normal and experimentally hydrated bovine corneas. Staining methods using cationic dyes were used for the detection.A 10 min fixation of cold microtome sections in absolute alcohol did not change the stainability of acid mucopolysaccharides substantially. The staining was only a little fainter (as against unfixed sections). After 10 min fixation with formol-cetylpyridinium chloride the staining of sections was diminished and after 30 min fixation in this fluid completely abolished. After formol-calcium chloride fixative the staining was decreased in dependence on the time of fixation due to the elution of acid mucopolysaccharides in the fixative (acid mucopolysaccharides in the fixative were demonstrated by means of paper electrophoresis). Formolcalcium chloride is likewise unsuitable.Experimental hydration of corneas in distilled water did not substantially alter the staining properties of acid mucopolysaccharides in cold microtome sections. Only quantitative differences were found in comparison with untreated corneas. These differences were due to hydration causing an increase in the distance of acidic groups among individual molecules of acid mucopolysaccharides.     

Extractability of glycoproteins and mucopolysaccharides of brain

Very little is known about the localization and functions of the glycoproteins and mucopolysaccharides of nervous tissue. There have been two major approaches to the study of these substances in brain. The first involves the isolation of glycopeptides and mucopolysaccharides after digestion of the lipid-free protein residue from whole brain with proteolytic enzymes (Margolis , 1967; Di Benedetta et al., 1969; Margolis and Margolis , 1970; Katzman , 1972). This approach has the advantage that sufficient tissue is used to permit analysis of the structure and metabolism of the carbohydrate components of these macromolecules. However, any differentiation of the various glycoproteins and mucopolysaccharides based on such features as their anatomical location, association with proteins, lipids or other membrane components, and the properties conferred by their non-carbohydrate portion, is unavoidably lost as a consequence of the procedures used for their isolation. On the other hand, several laboratories have attempted to study the glycoproteins and mucopolysaccharides of nervous tissue by treating brain (or subcellular fractions) with various detergents, and then examining the extracts for the pattern of separation obtained by polyacrylamide gel electrophoresis in terms of carbohydrate staining reactions or the incorporation of labelled precursors (Bosmann , Case and Shea , 1970; Duiton and Barondes , 1970; Quarles and Brady , 1971; Waehneldt , Morgan and Gombos , 1971). This approach has the advantages of relatively high sensitivity and the ability to study intact glycoproteins rather than glycopeptides produced by proteolytic enzyme digestion. However, it is presently impossible to identify any of the numerous and often poorly resolved bands thus obtained with glycoproteins or mucopolysaccharides of known structure and chemical composition, or in many cases even to identify the various complex carbohydrates as being glycoproteins, glycolipids or acid mucopolysaccharides. In an attempt to obtain some indication of the degree of anatomical heterogeneity of these compounds in nervous tissue, we have sequentially treated whole rat brain with several solvents to obtain intact glycoproteins and mucopolysaccharides. After removal of lipids and digestion with pronase, the composition of the glycopeptides and mucopolysaccharides has been analyzed.     

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.     

Enzymatic Elimination of Collagen in the Alcian Blue Staining of Connective Tissue Ground Substance

Collagenase pretreatment of frozen-dried sections permits Alcian blue staining of mucopolysaccharides of connective tissue ground substance without the interference of collagen staining. Hyaluronidase elimination of Alcian blue staining confirms mucopolysaccharide as a substrate of the staining reaction.     

Deamination to prevent the competitive effect of proteins in the metachromatic staining of sections at low pH-values

Summary To prevent the competitive cation effect of present proteins in the utilization of the effect of pH in differential histochemical staining of mucopolysaccharides with cationic dyes, preliminary oxidative deamination with 0.5% ninhydrin in absolute ethanol at 37° C was carried out. Thirty minutes deamination was sufficient to prevent the competitive effect of proteins at low pH values (1.0), even in tissue (cornea) with a high content of proteins compared with the content of acid mucopolysaccharides (glycosaminoglycuronoglycans).This work has been supported by grants from the Danish State Research Foundation.     

Some histochemical observations on the epididymis of rat--a comparison with chronological events in testis.

Histological and histochemical changes (lipids, phospholipids, neutral polysaccharides, acid mucopolysaccharides and sialic acid) were studied in the rat at pre- and postpubertal stages. At 10 days lipid and phospholipid staining was not observed both in the testis and epididymis though neutral and acid mucopolysaccharides and sialic acid were demonstrable. By 21 days, lipid and phospholipid staining was present in moderate amounts both in testis and epididymis. There was also a slight increase in other parameters studied. Maximum histochemical staining for all the parameters was seen at 60 days when the testicular and further components were well organized and functional. These findings reveal that both the testis and epididymis follow a similar pattern of development and are possibly governed by a common controlling factor--the androgens.     

Concanavalin A-perioxidase-diaminobenzidine (Con-A-PO-DAB)-alcian blue (AB): a reliable method for dual staining of complex carbohydrates.

A method has been established for the dual staining of complex carbohydrates in light microscopy. It is a combined concanavalin A-peroxidase-diaminobenzidine (Con-A-PO-DAB)-alcian blue (AB) (pH 2.5) method, and with this method it is possible to color alpha-D-glucosyl and alpha-D-mannosyl residues and acidic groupings of complex carbohydrates in tissues brown and blue respectively. Histochemical experiments using histological sections with reactive complex carbohydrates and casein films containing carbohydrates of known chemical structure have substantiated the validity of the above significance of the dual staining. Thus, the present dual staining method is a reliable one and a new addition to a series of dual staining techniques hitherto employed in the light microscopic histochemistry of complex carbohydrates.     

A New Pentachrome Method for the Simultaneous Staining of Collagen and Sulfated Mucopolysaccharides

Collagen is one of the most common fibers in the extracellular matrix, where sulfated mucopolysaccharides are also located. In addition, sulfated mucopolysaccharides are present in some globet cells and secretory glands. The objective of this article is to develop a new staining method that detects these two macromolecules simultaneously in the same sample. The method described stains tissues in five fundamental colors: collagen in red; sulfated mucopolysaccharides in violet; red blood cells in yellow; muscle in orange; and nuclei in green.As a conclusion, it will be interesting in the future to evaluate whether this method could be used as a basic histological method, as a histology teaching tool, or even in histopathological and cytopathological studies.     

Acid mucopolysaccharides in hair papillae of the stump-tailed macaque (Macaca speciosa).

Synopsis Acid mucopolysaccharides in dermal papillae of hair follicles from both bald and non-bald regions of the scalp of stump-tailed macaques were studied histochemically. Alcian Blue, Azure A and Periodic acid Schiff methods were used for staining mucopolysaccharides, and Bromphenol Blue for staining basic proteins. In an attempt to identify various polyanions, staining was carried out with Alcian Blue containing different concentrations of electrolytes. Methylation, saponification, mild acid hydrolysis and digestion with streptomyces or testicular hyaluronidase, chondroitinase ABC, or sialidase, were also used. The results indicate that chondroitin sulphate B is present in the papillae of terminal hair follicles in early and intermediate anagen, and degraded chondroitin sulphates are present in the papillae of vellus and terminal hair follicles in late anagen.     

The Reactions of Isolated Mucopolysaccharides to Several Histochemical Tests

The reaction to three histochemical tests of preparations of hyaluronic acid, chondroitin sulfate, heparin, the acidic mucopolysaccharides from cornea, gastric mucin, and dentine, and also of the neutral mucopolysaccharide from gastric mucin was studied. To 1% aqueous solutions of the acid mucopolysaccharides, equal volumes of 1% casein solution were added; drops of the resulting solutions were placed on slides and dried at 37 °C. The films were then fixed in acetic-alcohol (1:9). The technics employed were the periodic acid-Schiff (PAS) test, the metachromatic reaction and the Hale test. The relative acidity of the preparations was demonstrated by staining in dilute aqueous methylene blue at pH 3-6. With the exception of the preparation from dentine, the acid mucopolysaccharides stained only weakly with PAS; the neutral mucopolysaccharide stained strongly. It is concluded, therefore, that the use of the PAS technic for the histochemical demonstration of acid mucopolysaccharides is misleading, for many important members of this class of tissue component do not react appreciably. On the other hand, metachromasia was shown by all the acidic compounds studied, and the intensity of staining was approximately correlated with the acidity of the preparations. The Hale method was found to be nonspecific.     

The use of double staining techniques for investigating bacterial attachment to mucopolysaccharide-coated epithelial cells

Double staining techniques were devised to study Escherichia coli attachment to mucopolysaccharide-coated urinary tract epithelial cells. In addition, vital stains were used to distinguish between viable and nonviable epithelial cells. Alcian blue was used to confirm the presence of glycosaminoglycans and periodic acid Schiff was used to distinguish a further group of polysaccharides, proteoglycans, neutral mucosubstances and glycolipids. The staining methods enabled investigations to be carried out concerning the possible importance of mucopolysaccharides in the attachment of bacteria to mucosal surfaces. Staining techniques were also used to investigate whether the presence of a mucopolysaccharide coat is related to epithelial cell viability. The combinations of vital and mucopolysaccharide stains were found to give reproducible results when cell preparations were evaluated by three individuals. Both in vivo and in vitro certain populations of epithelial cells have been found with a large number of bacteria preferentially attached. The double staining techniques described here may help to reveal the nature of these target cells.     

Interactions of sulfated mucopolysaccharides with lectins. Application to the separation of mucopolysaccharide mixtures.

The interaction of sulfated mucopolysaccharides and lectins has been studied by determining the amount of precipitate formed when mucopolysaccharides are added to a solution of concanavalin A or a partially purified lectin preparation from red kidney bean (Phaseolus vulgaris). The amount of insoluble complex obtained when a given mucopolysaccharide is added to a solution of partially purified red kidney bean preparation is pH dependent. The reaction of concanavalin A and heparin has also been studied by adding increasing amounts of mucopolysaccharide to a fixed amount of lectin. This interaction results in the development of a precipitin-like curve and leads to the isolation of a heparin fraction which has been found to be more reactive with respect to formation of a precipitate than the original heparin preparation. Monosaccharides such as α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine which are known to bind specifically to the lectin, greatly inhibit precipitate formation. The interactions between sulfated mucopolysaccharides and lectins have been used to isolate various sulfated mucopolysaccharides.     

The Use of Double Staining Techniques for Investigating Bacterial Attachment to Mucopolysaccharide-Coated Epithelial Cells

Double staining techniques were devised to study Escherichia coli attachment to mucopolysaccharide-coated urinary tract epithelial cells. In addition, vital stains were used to distinguish between viable and nonviable epithelial cells. Alcian blue was used to confirm the presence of glycosaminoglycans and periodic acid Schiff was used to distinguish a further group of polysaccharides, proteoglycans, neutral mucosubstances and glycolipids. The staining methods enabled investigations to be carried out concerning the possible importance of mucopolysaccharides in the attachment of bacteria to mucosal surfaces. Staining techniques were also used to investigate whether the presence of a mucopolysaccharide coat is related to epithelial cell viability. The combinations of vital and mucopolysaccharide stains were found to give reproducible results when cell preparations were evaluated by three individuals. Both in vivo and in vitro certain populations of epithelial cells have been found with a large number of bacteria preferentially attached. The double staining techniques described here may help to reveal the nature of these target cells.     

Histochemistry and ultrastructure of the metacercarial cysts of blackspot trematodes Uvulifer ambloplitis and Neascus pyriformis

Cysts of Uvulifer ambloplitis from green sunfish, Lepomis cyanellus, and Neascus pyriformis from red shiners, Notropis lutrensis, were studied with light-level histochemistry and scanning and transmission electron microscopy. Cysts of both species are bilayered, consisting of an outer host capsule and an inner parasite cyst; the space between these layers is filled with a viscous material. The outer portion of the host capsule of both species is composed of fibrocytes, melanin granules, and collagen fibrils, and the inner portion of layers of flattened fibrocytes. The parasite cyst of U. ambloplitis is formed of 2 layers, an outer dense layer and an inner light layer, whereas the parasite cyst of N. pyriformis is made of 3 layers. A thin outer light-staining layer is present in addition to the 2 layers observed in U. ambloplitis. Results of histochemical staining were the same for both species. The host capsule stained positively for proteins and neutral and acid mucopolysaccharides. The viscous material was positive for neutral and acid mucopolysaccharides but not for proteins. The parasite cyst gave a strong positive reaction for neutral mucopolysaccharides but was negative for acid mucopolysaccharides and proteins.     

Morphology and histochemistry of the caudal gland of Corynopoma riisei Gill

Males of the South American freshwater fish, Corynopoma riisei , have glandular masses on the caudal peduncle and fin which are partly enclosed by a scale-covered pouch. Histo-chemical examination reveals an extensive layer of large secretory cells superficial to the dermis appearing similar in form to typical mucous cells. PAS and Alcian blue staining indicate all cells produce muco- and/or glycoproteins and that a few cells are also synthesizing mucopolysaccharides. This secretion probably acts as a pheromone during courtship to stimulate or release female sexual response.     

Histochemical differentiation of glycoconjugates occurring in the tilapine intestine

The glycoconjugates secreted by the anterior and posterior intestinal segments of Tilapia spp. were characterized by means of both conventional histochemical procedures (PAS, AB, AB-PAS, LID, HID) and a battery of 12 horseradish peroxidase labelled lectins. Some sections were treated with glycolytic enzymes and KOH before conventional and lectin stainings. The large majority of the mucopolysaccharides secreted by the goblet cells of the anterior segment are carboxylated while only a few carry sulphate groups. The mucopolysaccharides in the anterior intestine contained chondroitin, heparin and chondroitinsulphates which can provide protection for intestinal mucosae. DBA lectin staining demonstrated the presence of some endocrine cells in the anterior segment.     

Histochemistry of the glands associated with the reproductive tract ofLymnaea stagnalis

Summary The albumen gland, the muciparous gland and the oöthecal gland of female genital tract of Lymnaea stagnalis, collected in spring, autumn and winter have been studied.The reactions for polysaccharides, proteins and RNA have been performed in order to characterise the secretion of the glands.The albumen gland secretion consists almost exclusively of slightly acid polysaccharides whose histochemical reactions, according to Lison and Grainger and Shillitoe confirm the presence of galactogen. Proteins are also present in the secretion. The muciparous gland secretion consists of strongly acid mucopolysaccharides (non sulphated) produced by large cells among which small cells containing sulphated mucopolysaccharides are present.In the oöthecal gland two zones are present, one with a single type of cells containing acid mucopolysaccharides, and the other with two different types of cells: the first with mucopolysaccharides and the second with sulphated mucopolysaccharides, proteins and glycogen at the basis of the cell. Sialic acids are not present in the secretion of the glands studied.The polysaccharidic composition of the secretion of the glands is different from gland to gland. The secretion of the glands gradually changes and gets acid according to the composition of the various membranes and envelopes wrapping up the eggs.