A new histochemical method for the selective periodate oxidation of total tissue sialic acids

Summary Evaluation of the intensity of the periodic acid—Schiff (PAS) staining produced following oxidation for 1 h at 4°C with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that this reagent completely oxidized all available sialic acid residues of either the sialo- or sialosulphoglycoproteins of human and rat colon or the sialoglycoproteins of rat sublingual gland. These conditions produced no visible Schiff staining of either neutral macromolecules orvicinal diols located on hexose, 6-deoxyhexose orN-acetylhexosamine residues (neutral sugars) of sialo- and sialosulphoglycoproteins. Furthermore, there was no extraction of epithelial glycoproteins or de-O-acylation of side chain substituted sialic acid residues. These data demonstrate that 0.4mm periodic acid in approximately 1m hydrochloric acid can be used as a specific reagent for the selective visualization of sialic acids in the PAS procedure.Studies of the mechanism of the oxidation of neutral sugars with 0.4mm periodic acid in approximately 1m hydrochloric acid indicated that their lack of PAS reactivity was not due to the production of Schiff unreactive hemiacetals or hemialdals. It is suggested that the selectivity of 0.4mm periodic acid in approximately 1m hydrochloric acid is a result of an increase in the rate of the oxidation of the sialic acid residues together with a decrease in the rate of oxidation of neutral sugars.     

Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulphate ester. I. Methods based upon the selective periodate oxidation of sialic acids

Summary Five new methods, based upon the selective oxidation of sialic acid residues with 0.4mm periodic acid in approximately 1m hydrochloric acid at 4°C for 1 h (PA^*), have been devised for the simultaneous visualization of neutral sugars and either sialic acid and its side chainO-acyl variants orO-sulphate ester. In the first of these, the selective periodate oxidation—borohydride reduction—saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (PA^*—Bh—KOH—PA^*—T—KOH—Bh—PAS) technique, sialic acids withO-acyl substituents at C7, C8 or C9 (or which have two of three side chainO-acyl substituents) stain blue while neutral sugars with periodate-sensitivevicinal diols (hexose, 6-deoxyhexose, andN-acetylhexosamine) stain magenta. The second method, the saponification—selective periodate oxidation—Thionin Schiff—saponification—borohydride reduction—periodic acid—Schiff (KOH—PA^*—T—KOH—Bh—PAS), stains all sialic acids blue and neutral sugars magenta. In the third procedure, the selective periodate oxidation—Thionin Schiff—borohydride reduction—periodic acid—Schiff—saponification (PA^*—T—Bh—PAS—KOH) method, sialic acids without side chain substituents (or which have anO-acyl substituent at C7) stain blue and neutral sugars stain magenta. In the fourth method, the saponification-selective periodate oxidation—borohydride reduction—Alcian Blue pH 1.0—periodic acid—Schiff (KOH—PA^*—Bh—AB1.0—PAS) technique,O-sulphate esters stain aquamarine blue and neutral sugars stain magenta. In all of these techniques mixtures of the components stain in various shades of purple. Performance of the KOH—PA^*—Bh—AB1.0—PAS technique without the Alcian Blue pH 1.0 step provides a method for the selective identification of neutral sugars in macromolecules that also contain sialic acids.     

Periodate oxidation studies in the elucidation of the structures of sialic acid containing oligosaccharides

The structures of sialo-oligosaccharide alditols as determined by ¹H-NMR spectrometry together with methylation analysis did not correspond with those derived previously from quantitative periodate oxidation data alone. Possible causes of the discrepancy were explored in the periodate oxidation methodology. No free sialic acid was released by the acidity of the periodate in the course of oxidation at pH 4.5. The anionic properties of the sialic acid residues were therefore utilized to separate the periodate oxidation products and thereby establish the position of the sialic acid in the oligosaccharide chain.     

Binding Patterns of Lectins with GalNAc specificity in the mouse dorsal root ganglia and spinal cord

To localize membrane glycoconjugates in neurons of the mouse spinal cord and dorsal root ganglia (DRG), cryostat sections of newborn (P0), 7 day-old (P7), P14, P21 and P31 animals were stained with ten FITC-conjugated plant lectins, the majority of them recognizing N-acetyl-D-galactosamine (GalNAc) terminal sugar residues. In the dorsal root ganglia of P0 animals, the different lectins showed distinct patterns of labeling in either cells of the nervous system, including neurons, or other structures such as nerves or blood vessels. Moreover, some of these lectins showed important changes in their pattern of labeling during postnatal development. This was especially relevant for lectins that label a subpopulation of small-sized cells that have been previously identified as the nociceptive cells of the DRG. Enzymatic digestion of sections with neuraminidase removes sialic acid from the carbohydrate chains of glycoconjugates thus exposing novel sugar residues. When this treatment was applied to DRG sections from postnatal animals the pattern of lectin staining was either changed or eliminated and heterogeneous subsets of glycoconjugates normally masked by this sugar were exposed. In the spinal cord of PO animals, none of the lectins labeled cells in the central gray matter. However, after the enzymatic digestion of sections with neuraminidase, spinal cord motoneurons and some other cells were labeled by two of the lectins suggesting that GalNAc residues present in these cells are normally masked by terminal sialic acid. Altogether, these results show important changes in the temporal and spatial expression of glycoconjugates that may be relevant for the postnatal development of the CNS and PNS of mice.     

Light and electron microscopic demonstration of sialic acid residues with the lectin from Limax flavus: a cytochemical affinity technique with the use of fetuin-gold complexes

The development of a cytochemical affinity technique for the demonstration of sialic acid residues by light and electron microscopy is reported. The lectin from the slug Limax flavus, with its narrow specificity for N-acetyl- and N-glycolylneuraminic acid, was applied to tissue sections. Subsequently fetuin-gold complexes were used to visualize the tissue-bound lectin. Different cytochemical controls, including sugar inhibition tests, neuraminidase digestion, the use of fetuin-gold complexes alone, or acid hydrolysis of sections, proved the specificity of the technique. Postembedding staining was performed on frozen, paraffin, or semithin resin sections for light microscopy and on thin sections from low temperature Lowicryl K4M-embedded material for electron microscopy. The distribution of sialic acid residues in rat pancreas, liver, and colonic mucosa was investigated.     

KDN (Deaminated neuraminic acid): Dreamful past and exciting future of the newest member of the sialic acid family

KDN is an abbreviation for 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, and its natural occurrence was revealed in 1986 by a research group including the present authors. Since sialic acid was used as a synonym for N-acylneuraminic acid at that time, there was an argument if this deaminated neuraminic acid belongs to the family of sialic acids. In this review, we describe the 20 years history of studies on KDN (KDNology), through which KDN has established its position as a distinct member of the sialic acid family. These studies have clarified that: (1) KDN occurs widely among vertebrates and bacteria similar to the occurrence of the more common sialic acid, N-acetylneuraminic acid (Neu5Ac), but its abundant occurrence in animals is limited to lower vertebrates. (2) KDN is found in almost all types of glycoconjugates, including glycolipids, glycoproteins and capsular polysaccharides. (3) KDN residues are linked to almost all glycan structures in place of Neu5Ac. All linkage types known for Neu5Ac; α2,3-, α2,4-, α2,6-, and α2,8- are also found for KDN. (4) KDN is biosynthesized de novo using mannose as a precursor sugar, which is activated to CMP-KDN and transferred to acceptor sugar residues. These reactions are catalyzed by enzymes, some of which preferably recognize KDN, but many others prefer Neu5Ac to KDN. In addition to these basic findings, elevated expression of KDN was found in fetal human red blood cells compared with adult red blood cells, and ovarian tumor tissues compared with normal controls. KDNase, an enzyme which specifically cleaves KDN-linkages, was discovered in a bacterium and monoclonal antibodies that specifically recognize KDN residues in KDNα2,3-Gal- and KDNα2,8-KDN-linkages have been developed. These have been used for identification of KDN-containing molecules. Based on past basic studies and variety of findings, future perspective of KDNology is presented.     

Interaction between Na+-dependent transport systems for sugars and amino acids. Evidence against a role for the sodium gradient

Summary The concept that interaction between sodium-dependent transport systems represents competition for energy inherent in the transmembrane sodium gradient was examined with the use of isolated intestinal epithelial cells. The isolated cells exhibit transport interactions which are more significant in magnitude than those which have been described for intact tissue preparations. Accumulation of 1mm valine is inhibited 60% by 10mm 3-OMG. Conversely, uptake of 1mm 3-OMG is inhibited only 20% by 10mm valine. These data suggest that 3-OMG must discharge the cellular Na⁺ gradient more effectively than valine, if Na⁺ gradient dissipation can be taken as a basis for the inhibitory interaction. However, entry of 10mm 3-OMG is significantly slower than the entry of 10mm valine. Even if appropriate corrections are made for passive substrate entry and for differences in Na/substrate entry stoichiometry, it appears that valine should be somewhat more effective than 3-OMG in discharging the Na⁺ gradient. In light of these facts, it seems unlikely that the mechanistic basis for interaction between sugar and amino acid transport systems can be related to concomitant co-entry of Na⁺. It is suggested that the interaction results instead from competition for energized intermediates generated at limited rates by basic energy transduction events associated with the cell membrane which serve in support of a variety of active transport systems.     

Distribution of sugar residues in the bovine testis during postnatal ontogenesis demonstrated with lectin-horseradish peroxidase conjugates

Summary In the present study the distribution of various sugar residues in the cells of the male gonad during postnatal organogenesis was examined employing eight lectin-horseradish peroxidase conjugates (BS-I, ConA, DBA, PNA, RCA-I, SBA, UEA-I, WGA) on paraffin-embedded testicular tissue. The tissue was obtained from bull calves and young bulls of recorded age (4, 8, 16, 20, 25, 30, 40 and 52 weeks) and two adult bulls. During the whole observation period, lectin affinity in the developing testicular tubules was restricted to the germ cell line, while the Sertoli cells and their precursors remained completely unstained. DBA, a lectin with specific affinity to -d-GalNAc, served as a selective marker for prespermatogonia (PSG), the only precursors of bovine spermatogonia until the onset of spermatogenesis at week 30. -d-GalNAc, detected in the PSG Golgi zone and its vicinity, seems to play an important role during PSG proliferation and migration in the prepuberal testis. Concomitant with the differentiation of PSG into spermatogonia, the binding intensity of DBA to the Golgi zone of these cells decreased. After the gradual onset of spermatogenesis, the lectins revealed staining of Golgi complexes of most germ cell stages. Glycosylation of the cell components takes place in the Golgi complex, which explains the strong affinity of the lectins to this cell compartment. Inner and outer membrane of the acrosomal complex of spermatids, especially during Golgi and cap phase of spermiogenesis, were intensely stained with PNA, RCA-I and SBA. This staining disappeared in the maturation phase at the latest and indicates a role of terminal d-Gal-(13)-d-GalNAc, d-Gal and d-GalNAc during the formation of the sperm head and intraepithelial orientation of the spermatid. Other parts of the spermatid, such as the anulus and the cytoplasmic droplet, exhibited d-Gal, d-GlcNAc or sialic acid and d-GalNAc. In the intertubular tissue BS-I, RCA-I and UEA-I bound to vascular endothelia. Components of the intertubular extracellular matrix were stained with ConA (-d-Man), RCA-I (d-Gal), UEA-I (-l-Fuc) and WGA (d-GlcNAc or sialic acid).     

Histochemical characteristics of glycoproteins in the bile duct system of mice immunized with swine serum

Summary The bile duct system of BALB/c and DDY mice, which were immunized with swine serum (SS) or not, was examined histochemically. Biliary epithelial cells of the SS-treated BALB/c mice, which were positively stained with periodic acid-Schiff (PAS) and had binding sites of Dolichos biflorus (DBA), were thought to secrete neutral glycoproteins with terminal N-acetyl-d-galactosamine residues. Those of the SS-treated DDY mice were however negatively or weakly stained with any histochemical stainings. On the other hand, glandular epithelial cells of the SS-treated mice of both strains, which were positively stained with high iron diamine-alcian blue (HID-AB) and had binding sites of DBA, Griffonia simplicifolia-II (GS-II), Ulex europaeus-I (UEA-I), and Triticum vulgaris (WGA), were thought to secrete glycoproteins with terminal sialic acid residues. Biliary and glandular epithelial cells of the normal mice contained only a small amount of glycoproteins showing similar histochemical characteristics to those in the SS-treated BALB/c mice. BALB/c mice immunized with SS were thought to be very useful for the investigation of production and secretion of glycoproteins in the bile duct system as well as being good model of bile duct disease.     

Some quantitative histochemical studies of the periodic acid oxidation of glycogen in situ

Synposis The periodic acid oxidation of the glycogens in sections of ox liver fixed in Carnoy's fluid has been studied quantitatively. It was found that in 0.02 M acetate buffer at pH 5.0 the oxidation was rapid at first but levelled off after I hr. Even after prolonged oxidation (12 days), not more than 23–24% of the total available glycogen was oxidized. However, in the presence of electrolytes (e.g. 0.2.m sodium chloride) the oxidation was much greater. After 2.5 hr, for example, 40% of the available glycogen was oxidized. There was little difference in the velocity of oxidation in sections mounted on glass-slides and free-floating ones, or in free-floating sections of different thicknesses.Mounted and unmounted sections consumed on average 11–17 times more periodate than could be accounted for by the oxidation of their glycogen content.The results are interpreted in terms of a complex formed between periodate ions and the cuter glucosyl residues of the glycogen aggregate. The negative charge on the complex, it is suggested, prevents free periodate ions approaching and oxidizing the inner glycosyl residues.     

Histochemical characterization of glycoconjugates in the epithelium of the extrapulmonary airways of several vertebrates

Summary The glycoconjugates of the extrapulmonary airways of 11 tetrapode vertebrates have been characterized by means of both conventional and lectin histochemistry. Abundant sialosulphomucins were detected in the secretory cells and periciliary layer of turtles, snakes, birds and mammals while only sialomucins were observed in amphibians. Neutral and traces of acidic mucins were detected in the secretory cells of lizards. The secretory cells of the amphibian airways were reactive to Con-A, DBA and WGA. No -l-fucose residues reactive with UEA-I or LTA were detected in amphibians. The goblet cells of the turtles were stained by DBA, SBA and WGA. Secretory cells of snakes and lizards reacted with Con-A and WGA. The mucous goblet cells of the birds were reactive to Con-A, LTA and WGA. In the chicken, they also showed affinity for PNA and SBA. The ciliated cells ofthe avian species studied were stained by Con-A and WGA. Mammalian goblet cells were reactive to Con-A, UEA-I and WGA. In the rat, affinity for DBA and SBA was also observed. The present results reveal the existence of marked differences in the sugar residues of the glycoconjugates of the extrapulmonary airways of tetrapode vertebrates. Only sialic acid residues appear to be constant constituents of the glycoconjugates of the airways of all species studied.     

Spin-labelling of sialic acid in soluble and cell surface glycoproteins

A new, mild method is described for spin-labelling sialic acid residues in situ. The procedure involves the formation of C-1 sialamides and has been applied to a serum glycoprotein, a mucin, tissue sections from human colon, and erythrocyte membrane components. The selectivity of the method and its possible applicability to other types of labelling are discussed.     

Differential distribution of lectin-binding glycoconjugates in the secretory granules of hamster oviductal ampulla during the estrous cycle: a quantitative cytochemical analysis

 High resolution lectin-gold cytochemistry was used to quantitatively analyze the distribution of glycoconjugates in the hamster oviductal ampulla during the five stages of the estrous cycle. Lectins binding to N-acetyl-d-galactosamine-, d-galactose-, and sialic acid-associated glycoconjugates in the secretory granules of ampullary epithelial secretory cells showed staining of equal intensity throughout the five different stages of the estrous cycle. In contrast, the labeling intensity of glycoconjugates which contain N-acetylglucosamine as terminal sugar residues reached its maximum around the time of ovulation, i.e., at proestrus. Glycoconjugates which carry fucose and mannose as terminal sugar residues appeared to be totally absent from the secretory granules of the oviductal ampulla during the estrous cycle. Together, electron microscopic observations combined with quantitative results indicate that N-acetyl-d-galactosamine-, d-galactose-, and sialic acid-associated glycoconjugates may be secreted into the ampullary lumen irrespective of the stage of the estrous cycle, whereas the secretion of certain N-acetylglucosamine-associated glycoconjugates is stage specific and reaches its peak at the time of ovulation. These findings suggest that, at the time of ovulation, the ampullary epithelium changes its secretory activity and contributes its secretory products to the zona pellucida of oocytes freshly released from the ovary. Accepted: 10 August 1998     

Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranitro BT method

Summary The indoxyl—tetranitro BT method for the demonstration of alkaline phosphatase activity has been optimized and its validity for quantitative histochemistry tested. The study has been performed with model films of polyacrylamide gel incorporating homogenate of rat liver and with cryostat sections from the same livers. Addition of polyvinyl alcohol to the incubation medium greatly improved the localization of the final reaction product in cryostat sections. In polyacrylamide films, the formazan production specifically due to alkaline phosphatase was highest when using a medium containing 100mm Tris-HCl buffer, pH 9.0, 0.2–1.0mm substrate, 0.32mm 1-methoxyphenazine methosulphate, 10mm MgCl₂, 5mm sodium azide and 1mm tetranitro BT. For the incubation of cryostat sections in the presence of polyvinyl alcohol, the same medium could be used but the optimum concentrations of substrate and tetranitro BT appeared to be 1–2mm and 5mm respectively. The test minus control reaction was specific for alkaline phosphatase activity and could be inhibited completely with tetramisole. The test minus control reaction was linear with time up to 30 min with model films and up to 15 min with cryostat sections. The formazan production was also linear with the amount of homogenate incorporated in model films and with section thickness up to 18 µm and therefore, the reaction obeyed the Beer—Lambert law. Variation of the substrate concentration yielded aK _M of 0.05mm for aqueous media and aK _M of 0.55mm for polyvinyl alcohol-containing media. The inhibition with tetramisole appeared to be competitive withK _i = 0.07mm for aqueous media andK _i = 0.7mm for polyvinyl alcohol-containing media. These values indicate that the indoxyl—tetranitro BT method is considerably more sensitive than any metal salt or diazonium salt method developed so far. It is concluded that the optimized method described here is a specific, sensitive and valid quantitative histochemical method for the demonstration of alkaline phosphatase activity.     

LOCALIZATION AND IDENTIFICATION OF GALACTOSE/N-ACETYLGALACTOSAMINE AND SIALIC ACID-CONTAINING PROTEINS IN CHINESE HAMSTER METAPHASE CHROMOSOMES

Four lectins were used to recognize galactose/N-acetyl-galactosamine (Gal/GalNAc) and sialic acid residues in proteins of Chinese hamster metaphase chromosomes. In situ binding pattern of a fluorescein isothiocyanate-labelled (Gal/GalNAc)-specific lectin Sophora japonica agglutinin (SJA) showed that chromosomal SJA-binding proteins are primarily localized to the helically coiled substructure of chromatids. Numerous SJA-binding proteins were identified in Western blots of chromosomal proteins, their molecular weights ranging from 26 to 200kDa. Another Gal/GalNAc-specific lectin, peanut agglutinin (PNA), with a slightly different sugar binding specificity, did not bind to Chinese hamster metaphase chromosomes, and in Western blots only two chromosomal protein bands were faintly stained. The in situ labelling patterns of two sialic acid-specific lectins, Maackia amurensis (MAA) and Sambucus nigra (SNA) agglutinins, both showed that the helically coiled substructure of chromatids is also enriched in sialylated proteins. In Western blot analysis 11 MAA-binding protein bands with molecular weights ranging from 54 to 215kDa were identified, while SNA only bound to one protein band of 67kDa. MAA and SNA are specific for α (2|ad3)- and α (2|ad6)-linked sialic acid residues, respectively. Thus, it is likely that α (2|ad3)-linked sialic acid residues are more common in chromosomal proteins than α(2|ad6)-linked sialic acid residues. These data suggest that Gal/GalNAc and sialic acid-containing glycoproteins exist in metaphase chromosomes and that these proteins may have a role in the formation of higher order metaphase chromosome structures.     

Evaluation of the specificity of lectin binding to sections of plant tissue

Summary Hand sections of young corn root tips have been used in a study of problems encountered in the binding of fluorescently-labelled lectins to plant tissue. It was found, surprisingly, that with lectins specific for a sugar known to be present (Lotus andUlex lectins forl-fucose), with a lectin specific for a sugar thought not to be present (wheat-germ agglutinin for N-acetylglucosamine), with non-lectin glycoprotein and protein (-globulin and bovine serum albumin) and with basophilic dyes (alcian blue and toluidine blue), a coincidental binding pattern similar to the pattern of autofluorescence in the same tissue was obtained. Corn root tissues include cell walls composed of complex polysaccharides esterified with ferulic acid residues, as well as mucilages which are highly hydrated and expanded. In such material, neither standard inhibition controls with haptens nor the use of a wide range of lectin concentrations are adequate to distinguish clearly specific and non-specific binding of fluorescently-labelled lectin. Therefore, lectins are not the simple test probes they have been supposed. Before interpreting results obtained in using fluorescently-labelled lectins on any tissue sections, all available information (biochemical as well as histochemical) about the tissue must be considered.     

Masking of sperm maturation antigen by sialic acid in the epididymis of the mouse

Summary Sperm antigen expression during epididymal transit was examined in 4- to 16-week-old intact and castrated ICR mice, using the avidin-biotin complex (ABC) immunohistochemical method with monoclonal antibody T21 against a flagellar surface antigen. On untreated sections, the antigen was first expressed weakly on sperm in the proximal part of the corpus epididymis, and intraluminal components were stained in 4-week-old mice. Epididymal epithelial cells and their stereocilia, and cells in other reproductive organs were not stained. In contrast, on sections treated with neuramainidase, (1) the initial site of antigen appearance is a more proximal position in treated than in untreated sections, (2) stereocilia stained strongly, (3) the staining intensity of sperm and intraluminal components increased, and (4) some clear cells in the epithelium from the distal position of the caput to the corpus epididymis were stained. These results indicate that the antigen is produced by clear cells of the epididymal epithelium, that the antigenic determinant is masked initially by sialic acid residues, and that expression of the antigenic determinant on the sperm surface during epididymal maturation apparently involves desialylation.     

Further evidence by site-directed mutagenesis that conserved hydrophilic residues form a carbohydrate-binding site of human galectin-1

To identify critical amino acid residues for carbohydrate binding of galectins (soluble -galactoside-binding lectins found in the animal kingdom), site-directed mutagenesis was performed on human galectin-1. On the basis of the previous results (Hirabayashi and Kasai (1992)J Biol Chem 266:23648-53), more systematic mutagenesis experiments were performed in order to confirm the concept that conserved hydrophilic residues play a central role. When a homologous substitution was made for highly conserved His44, Arg48 or Asn61, the resultant mutant (H44Q, R48H or N61D, respectively) almost completely lacked carbohydrate-binding ability, as found previously for Asn46, Glu71 and Arg73 mutants. This suggests these six hydrophilic residues are essential. On the other hand, when less conserved Lys63, Arg111 or Asp125 were substituted, the resultant mutant (K63H, R111H or D125E, respectively) retained almost the same affinities to asialofetuin and lactose as the wild-type galectin. Therefore, none of these residues is directly involved in the binding. These results, together with the previous observation that the above six essential residues are all encoded in the largest exon of the gene and are located close to each other in the central, most hydrophilic region of the protein, suggest that the residues form a carbohydrate-binding site of galectin.Abbreviations EDTA-PBS 2mm EDTA, 20mm Na-phosphate, pH 7.2, 150mm NaCl - MEPBS EDTA-PBS containing 4mm -mercaptoethanol - IPTG isopropyl--(d)-thiogalactoside - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

A quantitative cytochemical assay for osteoclast acid phosphatase activity in foetal rat calvaria

Summary Acid phosphatase activity is prominent in osteoclasts (bone resorbing cells) and has been implicated in the process of bone resorption, although its precise role is not understood. To study the distribution and activity of this enzyme, a quantitative cytochemical method has been developed using undecalcified fresh frozen sections of foetal rat calvariae. Sections were allowed to react with 3mm naphthol ASBI phosphate at pH 5.0, and the reaction was stopped by rinsing in ice-cold tap water containing 50mm sodium fluoride. The reaction product was post-coupled to Fast Garnet at 4°C. The absorbance of areas of reaction product in the cytoplasm was measured using scanning and integrating microdensitometry. The initial velocity rate was maintained for up to 2 min at pH 5.0 with a substrate concentration of 3mm and a section thickness of 5 µm. Under these conditions reaction product was localized to osteoclasts and the surface of bone matrix beneath these cells. Activities in osteoblasts and chondrocytes were negligible. Osteoclastic acid phosphatase was almost totally inhibited by 10mm fluoride and reduced by 70% by 100mm tartrate.     

The effect of various fixation procedures on the digestability of sialomucins with neuraminidase

Synopsis The histochemical digestability with neuraminidase of sialomucin in mouse sublingual gland was studied in unfixed and formaldehyde vapour-fixed cryostat sections, and in sections prepared from paraffin-embedded material fixed in several alcohol- or formaldehyde-containing fixatives recommended for mucosubstances.The removal of sialic acid residues from sections treated with neuraminidase was followed histochemically with the following staining methods: Azure A pH 3.5, Alcian Blue pH 2.5, Low Iron Diamine and Alcian Blue pH 2.5 followed by periodic acid-Schiff. When Goland's methanolic cyanuric chloride was used as fixative, only a partial loss of tissue basophilia was evident after enzyme incubation, but in tissues fixed in other ways a complete loss of histochemically detectable sialic acid residues was observed.