The histochemistry of urea-unmasked glycosaminoglycans in the skin of the eel, Anguilla japonica

In the connective tissues of the dermis and subcutis of the eel skin, the histochemistry of urea-unmasked glycosaminoglycans has been studied by means of combined staining and enzyme digestion procedures. The staining procedures employed were alcian blue (AB) pH 1.0, AB pH 2.5, aldehyde fuchsin (AF), periodic acid-Schiff (PAS), AB pH 2.5-PAS, high iron diamine (HID) and low iron diamine (LID) methods, whereas the enzymes used were Streptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained have shown that a substantial amount of dermatan sulfate and a relatively small amount of hyaluronic acid, chondroitin, chondroitin sulfate A and/or C were the glycosaminoglycans involved in the connective tissues of the eel skin and that the tissues were devoid of keratan sulfate.     

Effects of Streptomyces hyaluronidase digestion on the histochemical reactions of proteoglycans in cartilage compared with its effects on certain non-cartilaginous tissues

Summary To test the value ofStreptomyces hyaluronidase in carbohydrate histochemistry, the effects of digestion with the enzyme on the staining of cartilage and non-cartilaginous tissues by Alcian Blue (AB) pH 1.0, AB pH 2.5, high iron diamine, low iron diamine, aldehyde fuchsin, dialysed iron-ferrocyanide and AB pH 2.5-periodic acid-Schiff were studied by light microscopy. The results obtained lead to the conclusion that theStreptomyces enzyme releases not only hyaluronic acid but also chondroitin sulphates and keratan sulphates in cartilage. Since hyaluronic acid is known to be linked to chondroitin sulphate proteoglycans, the enzyme is of limited value in localizing hyaluronic acid in cartilage. However, it is useful in localizing hyaluronic acid in most non-cartilaginous tissues.     

Insect mucosubstances. I. The mucosubstances of developing connective tissue in the locust,Locusta migratoria

Synopsis A layer of collagenous connective tissue develops around the ejaculatory duct of the male locust,Locusta migratoria, during the fourth and fifth instars and the first ten days of the adult stage. The mucosubstances associated with this tissue have been characterized by using a series of histochemical reactions, including Alcian Blue staining at different pH levels and salt concentrations the periodic acid-Schiff test and recent modifications of it, the high iron diamine test and enzymatic digestions. The results indicate that sulphated glycosaminoglycans accumulate during the developmental period, so that in the mature adult, the connective tissue probably contains chondroitin and dermatan sulphates, and possibly some keratan sulphate. Neutral glycoproteins also occur in the tissue.     

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. II. Human bronchial submucosal gland

Synopsis The effect of pH on Alcian Blue staining of sialomucins and sulphomucins in human bronchial submucosal glands has been analysed. Using Alcian Blue combined with periodic acid-Schiff, lowering the pH was associated with a decrease in the area staining with Alcian Blue and an increase in that staining with periodic acid-Schiff, save in one bronchus with a large sulphomucin content, in which an increase in the area staining with Alcian Blue was found at pH1.0. In all bronchi, an increase in the intensity of Alcian Blue staining was found at this pH. Sialomucin sensitive to sialidase was found to lose Alcian Blue staining at a higher pH than sialomucin resistant to the enzyme. Some sulphomucins stained with Alcian Blue throughout the pH range studied and some only at the more acid pH levels. At pH1.0 Alcian Blue stained only sulphomucins, thus distinguishing them from sialomucins. Alcian Blue staining combined with the high iron diamine technique has enabled three sulphate groups to be identified: one stained with high iron diamine, the other two did not, and, of the latter, one stained with Alcian Blue at pH 2.6 and1.0, and the other only at pH1.0.     

The effect of digestion with keratanase (Pseudomonas sp.) on certain histochemical reactions for glycosaminoglycans in cartilaginous and corneal tissues

Summary The effect of digestion with keratanase (Pseudomonas sp.) on the Alcian Blue (AB) pH 1.0, pH 2.5, Aldehyde Fuchsin, high iron diamine, low iron diamine and dialysed iron-ferrocyanide reactions has been tested in the costal and ear cartilage tissues of the rabbit and corneal tissues of the rat and rabbit. The effect of digestion with chondroitinases ABC and AC on the same reactions was examined in the same tissues for comparison. Digestion with keratanase diminished the intensity of all the reactions in the cartilage tissues to a variable extent; however, the diminutions in intensity of the reactions appeared to be less marked as compared with those following digestion with two chondroitinases. In the corneal stroma, all the reactions were markedly reduced in intensity following digestion with keratanase. In contrast, these reactions were only slightly or moderately diminished in intensity by digestion with the two chondroitinases. As glycosaminoglycans are known to be present in cartilage and corneal tissues and the substrate specificities of the three enzymes used are now well established, the present results are consistent with the concept that keratanase specifically degrades and releases keratan sulphates involved in the tissues.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Summary Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

Immunohistochemical staining for chondroitin sulphate and keratan sulphate. An evaluation of two monoclonal antibodies

Immunohistochemical staining with commercially available antibodies against chondroitin sulphate (clone CS-56) and keratan sulphate (clone 1/20/5-D-4) was compared with two conventional histochemical methods for the demonstration of glycosaminoglycans, namely Alcian Blue with varying pH and critical electrolyte concentrations, and a modified PAS stain. The antibodies were tested on sections from both frozen and fixed, paraffin embedded human material from umbilical cord, skin, and bronchus. The results showed immunostaining to function equally well on frozen and routine sections, and to be superior to Alcian Blue and PAS with regard to morphological detail. Thus, reactivity with anti-chondroitin sulphate was demonstrated in vessel walls, in small nerves, in the basal membrane zone of the skin, in perichondrium, and in and around chondrocytes. Reactivity with anti-keratan sulphate occurred in chondroid matrix and in perichondrial tissue; however, some cells of the bronchial epithelium and mucous glands also exhibited positivity.     

Insect mucosubstances. II. The mucosubstances of the central nervous system

Synopsis The glycosaminoglycans of the glial lacunar system and neural lamella of cockroach and locust ganglia have been characterized histochemically, using primarily Alcian Blue binding methods at various pH levels and salt concentrations, the periodic acid-Schiff test together with recent modifications, the high iron diamine test, and enzymatic digestions. The results suggest the presence of hyaluronic acid in the glial lacunar system and of a mixture of chondroitin and dermatan sulphates, together with keratan sulphate in the neural lamella. The significance of the presence of these substances in the central nervous system of insects is discussed.     

Sialic acid in colonic mucin: An evaluation of modified PAS reactions in single and combination histochemical procedures

Summary Two new histochemical procedures for detecting sulphated and non-sulphated sialomucin in colonic mucosa were assessed: the saponification—Alcian Blue pH 1—periodic acid—phenylhydrazine—Schiff method (KOH—AB pH 1—PAPS) and the mild periodic acid modification of this (KOH—AB pH 1—mPAS). Using normal colonic mucosa obtained from 11 non-cancer patients, the mPAS and PAPS techniques were tested for specificity and reproducibility for staining sialic acid, either alone or in combination with Alcian Blue. A spectrophotometric method was devised to quantify the uptake of both Schiff and Alcian Blue stain by sections. At low temperature and pH5.5, the mPAS procedure had improved specificity over the PAPS procedure, and after saponification it could be used to stainO-acetyl-substituted sialic acid. When used in combination with Alcian Blue at pH 1, however, underestimation of the sialic acid content occurred owing to interference between Alcian Blue and Schiff dyes. Interference was even greater with KOH—AB pH1—PAPS procedure for both sialic acid and sulphate components. We conclude that caution must be exercised in interpretation of the staining results obtained with these new combination methods and that more accurate information on the sialic acid and sulphate content of colonic mucin is obtained by staining serial sections with the mPAS technique and Alcian Blue pH 1 alone.     

The histochemical specificity of Streptomyces hyaluronidase and chondroitinase ABC.

Synopsis Treatment of tissue sections with enzymes which degrade specific types of glycosaminoglycans should provide a means for localizing glycosaminoglycans in tissue sections. The feasibility of this technique was examined by utilizing endogenously labelled glycosaminoglycans in chick and quail embryos. Less than 8% of the total glycosaminoglycans appear to be lost non-specifically during fixation and dehydration. BothStreptomyces hyaluronidase and chondroitinase ABC degraded more than 90% of their respective substrates and demonstrated minimal non-specific extraction of other glycosaminoglycans. The selectivity of chondroitinase ABC for sulphated glycosaminoglycans was substantially increased by raising the pH of the incubation buffer to 8.6. At this pH, chondroitinase ABC degraded negligible amounts of hyaluronic acid. Use of bothStreptomyces hyaluronidase and chondroitinase ABC confirmed that embryonic hyaluronic acid binds Alcian Blue under conditions that were previously believed specific for sulphated glycosaminoglycans. We suggest that this may be due to the increased molecular weight of embryonic hyaluronic acid compared to the hyaluronic acid in adult tissues. The results presented suggest that treatment of adjacent sections with buffer, chondroitinase ABC at pH 8.6, andStreptomyces hyaluronidase and subsequent staining with Alcian Blue provides a method for localizing and quantitating glycosaminoglycans in tissue sections.     

Histochemistry and morphology of porcine mast cells

Summary Mast cells have been described extensively in rodents and humans but not in pigs, and the objective of this study was to characterize porcine mast cells by histochemistry and electron microscopy. Carnoy's fluid proved to be a good fixative but fixation with neutral buffered formalin blocked staining of most mast cells. Alcian Blue stained more mast cells than did Toluidine Blue (pH 0.5), although Alcian Blue also stained goblet cells. In pigs, unlike rodents, the Alcian Blue method did not distinguish between mast cells in the intestinal mucosa and those in the connective tissue of the intestinal submucosa, tongue and skin. Mast cells were significantly larger in adult pigs than in piglets; in adult pigs and piglets, mast cells in the intestinal mucosa were significantly larger than those in submucosal connective tissue, and they were more varied in shape in piglets and adults. Granules in mast cells in the intestinal mucosa stained less intensely than those in mast cells in connective tissue of tongue, skin and intestinal submucosa. Mast cells in the connective tissue of the tongue, skin and intestinal submucosa fluoresced strongly when stained with berberine sulphate or with a mixture of berberine sulphate and Acridine Orange, but mast cells in the intestinal mucosa did not. All mast cells reacted positively in an enzyme-histochemical method previously used to detect human tryptase but not in a method previously used to detect human chymase. Mast cells in the medulla of thymus stained similarly to mast cells in the intestinal mucosa. Ultrastructural differences between mast cells were not detected.     

Ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland

Summary The ontogeny of sulphated glycoconjugate-producing cells in the rat fundic gland has been studied using high iron diamine (HID), Alcian Blue (AB) at pH 1.0, high iron diamine in combination with Alcian Blue at pH 2.5 (HID-AB), cationic colloidal gold (CCG) at pH 1.0 under light microscopy and CCG (1.0), HID-thiocarbohydrazide (TCH)-silver proteinate (SP)-physical development (PD) under electron microscopy. From day 19.5 of gestation, sulphated glycoconjugate-producing cells were discernible under both light and electron microscopy. The development of such cells can be classified into four stages: (1) a prenatal period from day 19.5 of gestation extending to 0.5 days after birth; (2) 1 day to 2 weeks after birth; (3) 2 to 4 weeks after birth; and (4) the final period from 4 to 8 weeks after birth. Glycoconjugate-producing cells reached maturity by 4 weeks after birth. Our results indicated that glycoconjugate-producing cells were cells along the wall of foveolar lumen, but not those covering the gastric mucosa surface. Our results also suggested that thetrans totransmost Golgi apparatus lamellae were the sites of sulphation in the developing rat stomach.     

Glycosaminoglycans in the touch corpuscle of the dog

Synopsis Alcian Blue staining of the touch corpuscle of dog skin indicates that glycosaminoglycans are present as globular masses in some of the basal cells and are also seen in high concentration in the connective tissue core. The function of these basal cells is not clear but they may be responsible for the production of the large amount of glycosaminoglycan present in the underlying dermis.     

The histochemical specificity of High Iron Diamine—Alcian Blue

Summary The specificity of the High Iron Diamine—Alcian Blue pH2.5 (HID—AB 2.5) procedure was examined in tissue sites containing sialogycoproteins alone or differing proportions of sialo- and sulphosialoglycoproteins. Studies with HID in differing final concentrations of hydrochloric acid or sodium chloride confirmed that staining is dependent upon both the pH and the ionic strength of the dye bath and demonstrated a marked heterogeneity in the pKa of the anionic groups of sialosulphoglycoproteins. Use of the sequence High Iron Diamine—Alcian Blue pH 1.0 demonstrated that complete or almost complete staining ofO-sulphate esters occurred when HID was prepared in water (final pH 1.3). However, under these conditions HID—AB 2.5 was shown to be non-specific because only black HID staining was observed in sites containing large quantities of sialic acids. This non-specificity was due either to the masking of Alcian Blue staining by HID and/or the black HID staining of anionic groups other than sulphate. These results may account for some of the conflicting data obtained by different groups of investigators who have studied transitional mucosa in human colonic diseases. Caution should be used in drawing conclusions from the use of HID—AB 2.5 without confirmatory evidence from other more specific procedures.     

Ultrastructure of the absorptive cell glycocalyx in hyperplastic colonic polyps after staining with Alcian Blue and high iron diamine

Summary The glycocalyx of absorptive cells in large intestinal hyperplastic polyp was characterized histochemically at the electron microscope level by the use of the Alcian Blue pH2.5 and high iron diamine techniques with the aim of comparing their ability in preserving the fine reticular network of the structure. Both the reagents stained glycocalyx, indicating the presence of sulphated acidic glycoconjugates. However, they showed different degrees of condensation of the reactive sites. Alcian Blue preserved its filamentous appearance better.     

Histochemical localization of protein-polysaccharides in renal tissue

The purpose of this study was to investigate the distribution of protein-polysaccharides in the glomerular and non-glomerular regions of the nephron. The techniques used include the digestion of kidney slices with specific polysaccharidases: neuraminidase, hyaluronidase, chondroitinase ABC, and collagenase followed by several cytochemical techniques to identify the glycosaminoglycans and glycoproteins at the light and electron microscope levels. Differential staining of hyaluronic acid and sulphated glycosaminoglycans was accomplished with Alcian Blue at pH 2.5 and pH 0.5, respectively. Sialoproteins were stained with Alcian Blue at pH 2.5. The periodic acid Schiff’s reaction technique was employed for the visualization of collagen. At the electron microscope level the polysaccharides were identified with the periodic acid-chromic acid-silver methenamine reaction. Our results indicated that the major polysaccharide components of the glomerular basement membrane were sialoproteins and collagen, with smaller amounts of hyaluronic acid and various sulphated glycosaminoglycans. Hyaluronidase digestion resulted in partial detachment of epithelial processes from the glomerular basement membrane indicating the hyaluronic acid may have a role in the stability of the attachment of these processes. Tubular basement membranes also contain sialoproteins and sulphated glycosaminoglycans but in considerably lower concentrations than the glomerular basement membrane. Bowman’s capsule appears to contain mostly sulphated glycosaminoglycans and has a lower concentration of sialoproteins and hyaluronic acid.     

Acid glycosaminoglycans of mouse neuroblastoma C1300 cells

Synopsis Cultured mouse neuroblastoma C1300 cells were examined for acid glycosaminoglycans using the Alcian Blue and periodic acid-Schiff staining techniques. It was found that the cells contained hyaluronidase-resistant sulphated glycosaminoglycans; hyaluronic acid, chondroitin sulphate, and sialoglycoproteins were not demonstrated. These properties are held in common with foetal mouse brain spongioblasts in culture. In contrast to the latter cells, but in common with some peripheral neuronsin vivo, C1300 cells were stained by the periodic acid-Schiff technique for neutral polysaccharides. The results are discussed in relation to the poor adhesive properties of neuroblastoma cells.     

The Alcian Blue and combined Alcian Blue-Safranin O staining of glycosaminoglycans studied in a model system and in mast cells

Synopsis Polyacrylamide films containing different glycosaminoglycans have been applied to the study of the Alcian Blue and combined Alcian Blue-Safranin O staining procedures. It was found that the polyacrylamide matrix can be interpreted as some kind of barrier around the substrate molecules, a situation which can be compared to a certain extent with what occursin situ, where complex protein molecules can likewise form a barrier.The Alcian Blue staining of the model films was found to follow the Lambert-Beer law. The time to reach optimal dye binding depended on the concentration of the glycosaminoglycan enclosed in the model films and on the concentration of Alcian Blue in the dye solution. Lowering the pH of the dye solution appeared to increase the rate of staining. Optimal staining of model films in the presence of salt or urea was not possible, because under these conditions the pores of the polyacrylamide matrix became blocked. Alcian Blue was found to bind irreversibly to the glycosaminoglycan molecules enclosed in the polyacrylamide films.The results of the combined Alcian Blue-Safranin O staining applied to model films appeared to be highly dependent on the amount of Alcian Blue bound to the glycosaminoglycan in the first step of the double staining procedure. No specific differences were noticed between the behaviour of the different glycosaminoglycan-Alcian Blue complexes towards the Safranin O binding in the mext step. As the theoretical basis for the application of the combined Alcian Blue-Safranin O staining was also found not to be completely valid, the conclusion was reached that this double staining cannot be used for the histochemical identification of glycosaminoglycans. The colour retained by a certain glycosaminoglycan-containing part of the specimen only delivers information about the accesibility of that part for Alcian Blue.     

Glycosaminoglycans of disaggregated foetal mouse brain tissue cultures

Synopsis Disaggregated foetal mouse brain tissue cultures were examined for glycosaminoglycans using Alcian Blue and periodic acid-Schiff staining techniques. It was found that spongioblasts (neuron and glial cell precursors) were rich in sulphated glycosaminoglycans, while astrocytes contained little or no sulphated polymers. The chief acid glycosaminoglycans of the brain reportedin vivo, hyaluronic acid, chondroitin sulphate and sialic acid-bearing polymers, were not demonstrated in the mouse brain cultures. There was a decline in glycosaminoglycan content over two weeks in culture, but during the corresponding periodin vivo an increase has been reported. These deficiencies are possibly correlated with the failure of the cultures to myelinate.     

Histochemical localization of acid mucopolysaccharide in the respiratory muscles of a fresh-water air-breathing siluroid fish, Clarias batrachus (Linn.).

Respiratory muscles involved in gill ventilation (= irrigation) of an amphibious siluroid fish, Clarias batrachus (Linn.) were studied by phase contrast and light microscopy after the treatment with PAS. Alcian Blue at pH 2.5 and 1.0, dialyzed iron and Toludine Blue. The transverse muscle bands lightly stained with PAS, Alcian Blue at pH 2.5 and 1.0 and Dialyzed Iron suggesting that the mucopolysaccharide occured in relatively low concentrations. Phase contrast microscopy indicated that the transverse bands stained by the above mentioned reagents correspond to the I-bands. Methylation for 4 hours at 60 degrees C prevented I-band staining with Alcian Blue in the muscles studied. Saponification alone left I-band alcianophilia intact. These findings reveal that myofibrillar I-bands of respiratory muscles contain sulphated acid mucosubstances.