Impurities and staining characteristics of Alcian Blue samples

Synopsis The composition of various samples of Alcian Blue^* and related dyes was studied, using t.l.c. (with cellulose as adsorbent andn-butanol: acetic acid: water as developing solvent), solvent extraction and precipitation, i.r. spectroscopy and classical semimicro analysis. All the Alcian Blue samples appeared to contain the same coloured components. The Alcian Green samples were mixtures of these blue components and Alcian Yellow. All the Astra Blue samples examined were composed of the same blue constituents. Colourless components identified were boricacid, dextrin and sulphate and sometimes amounted to nearly three-quarters by weight of the crude dyes. Impurities had only a slight effect on staining with Alcian Blue in aqueous acetic acid but appreciably affected staining by the critical electrolyte concentration (C.E.C.) procedure. Dextrin as impurity raised C.E.C. limits while the inorganic salt impurities raised the C.E.C. values of some substrates and lowered those of others.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

Microwave-assisted staining of mucosal mast cells and granulated intra-epithelial lymphocytes after formalin fixation

Summary Rat jejunum was fixed with either formalin or methanol-formalin acetic acid (MFAA) and stained with Astra Blue or Alcian Blue with or without microwave irradiation. Staining of both mucosal mast cells and granulated intra-epithelial lymphocytes after formalin fixation was considerably improved by microwave irradiation. On the other hand, microwave irradiation slightly impaired staining of mucosal mast cells (MMC) and even more strongly granulated intra-epithelial lymphocytes (GIEL) after MFAA fixation.     

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.     

The effect of pH on Alcian Blue staining of epithelial acid glycoproteins. I. Sialomucins and sulphomucins (singly or in simple combinations)

Synopsis This study is concerned with the staining of epithelial acid glycoproteins by Alcian Blue at various pH levels. A detailed analysis of the effect of pH on Alcian Blue staining of epithelial tissues at selected sites was made. Alcian Blue was combined with the periodic acid-Schiff technique, the Alcian Blue being used at pH levels between 2.6 and 0.5.Animal salivary glands, human foetal tracheal gland and epithelial goblet cells of the adult bronchial mucosa were chosen for study because the nature of their acid glycoprotein is known and is relatively simple.In sites containing sialomucin alone, no Alcian Blue staining took place below pH 1.5. A difference was demonstrated between sialidase-sensitive sialomucin which stained only between pH 2.6 and 1.7, and sialidase-resistant sialomucin which stained between pH 2.6 and 1.5. Two types of sulphomucin were identified: the usual one stained with Alcian Blue at all the pH levels studied, and the other, in the canine gland, stained only at the most acid pH levels, that is, between pH 1.5 and 0.5.     

The cytochemical localization of lysozyme activity in leucocytes

Synopsis A method for the cytochemical localization of lysozyme activity has been developed, using chitin as substrate. A homogenous film of substrate is prepared on a microscope slide by precipitation of chitin from a lithium iodide solution.Hydrolysis of the chitin substrate by lysozyme results in breakdown products which stain with Alcian Blue at pH 4. The method permits the cellular localization of lysozyme activity and has been applied to the neutrophils of peripheral blood which are known to be rich in lysozyme.     

Investigations of dye binding in the sequential staining of mucosaccharides by Alcian Yellow-Alcian Blue. II. Spot tests and experiments on dye-mucopolysaccharide (glycosaminoglycan) precipitates

Synopsis The amounts of dye bound to mucopolysaccharide in the histochemical sequential staining Alcian Yellow-Alcian Blue method were determined by studying dye-mucopolysaccharide (glycosaminoglycan) precipitates in test-tube and spot test experiments. In the second step (Alcian Blue) of the method, previously bound Alcian Yellow was released into the staining solution and simultaneously the precipitate took up Alcian Blue. The amounts of Alcian Yellow released and Alcian Blue taken up varied for different mucosaccharides, and depended on the staining time of the second step (Alcian Blue) of the sequence, as well as on the concentration of dye and salt in the Alcian Blue solution. It is thought that, among other things, the Alcian Blue in solution displaces some of the bound Alcian Yellow. Some observations could be explained by the aggregation of dye molecules. The results were in agreement with previous histochemical observations.     

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.     

Investigations of dye binding in the sequential staining of mucosaccharides by Alcian Yellow-Alcian Blue. 1. Histochemical experiments on different animal and human mucosaccharides

Synopsis It may be assumed that, histochemically, carboxyl groups and sulphate half-ester groups of muco electrolyte concentration of the dye baths in the two steps of a sequential Alcian Yellow-Alcian Blue method. In the present study the specificity and reliability of this method has been investigated.When the staining conditions were the same in both steps, the second dye (Alcian Blue) was found to stain mucosubstances in spite of the prior staining with Alcian Yellow. Binding of Alcian Blue was observed in all but very dilute Alcian Blue solutions. The degree of Alcian Blue binding depended on the dye concentration and staining time of the second step (Alcian Blue), and it varied widely for different mucosubstances. Although an incomplete saturation of anionic groups with dye molecules in the first step cannot be completely excluded, it is thought that Alcian Blue displaces Alcian Yellow from the carboxyl and sulphate groups of mucosubstances in tissue sections.It seems that the sequential Alcian Yellow-Alcian Blue method, under the conditions investigated, does not provide a reliable means for differentiating carboxyl and sulphate groups of mucosbstances in tissue sections simultaneously, because the second dye obviously is capable of displacing the first dye from sulphate groups. However, it is possible to distinguish non-sulphated acid mucosaccharides from sulphated mucosaccharides.     

Side effects of reaction media in histochemical blocking procedures

Synopsis 0.2 N NaOH, the reaction medium for 1,2-cyclohexanedione, a specific reagent for arginyl residues in proteins, was found to intensify, at some sites in rat abdominal skin and human gingiva, the Sakaguchi reaction, staining with Pauly's reagent, and anionic dye binding at pH 6.4; at other sites these reactions were reduced, presumably due to extraction of material from sections. 0.2 N NaOH slightly reduced staining after the ninhydrin-Schiff procedure at all sites in rat skin. The interpretation of this finding is obscure, because some sites giving a positive Sakaguchi reaction and staining with anionic dyes failed to stain after the ninhydrin-Schiff procedure. There were also alterations in staining, with the cationic dyes Alcian Blue and Alcian Yellow. It is suggested that 0.2 N NaOH ruptures linkages between polycationic residues of proteins and polanions, demonstrable by Alican Blue. The blockade produced by acetic anhydride-pyridine (4060 v/v) mixtures was stable, in the alkaline conditions required for staining with Pauly's reagent. Pretreatment with pyridine alone reduced tissue binding of anionic dyes.     

The cytochemical localization of lysozyme in Paneth cell granules

Synopsis The breakdown products resulting from the hydrolysis of chitin by lysozyme stain with Alcian Blue. A method based upon this observation has been developed for the histochemical demonstration of lysozyme activity. The application of this method to the jejunal crypts of several animal species indicates that Paneth cell granules contain lysozyme. The binding of the hydrolysis products with Alcian Blue is so strong that any other alcianophilia (e.g. of acid mucosubstances in goblet cells) can be removed selectively by washing withN-cetylpyridinium chloride and counterstaining with Basic Fuchsin and Nuclear Fast Red.     

Agglutination of the red blood cells after experimental glycolytic alteration]

The present findings proved Alcian Blue as hemagglutinin suitable for the specific demonstration of negatively charged sites of the cell surface. Erythrocytes with reduced surface charge because of previous trypsination or desialization exhibited decreased Alcian Blue agglutination. On the other hand, Alcian Blue agglutinates more intensely red blood cells with a high surface charge (reticulocytes). Findings with A antiserum or the lectins prepared from Lens culinaris and Helix pomatia indicate a masking of glycolipidic group A receptors by glycoproteins of the erythrocyte membrane. Effects of enzymatic degradation (neuraminidase, trpysin, pronase) combined with incubation in NaF-medium for the deprivation of ATP could be explained by a rearrangement of receptors resulting in altered agglutinability of erythrocytes.     

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.     

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.     

Histochemistry of protein-bound disulphide groups in the duct secretory granules of the rat submandibular gland

Synopsis The existence of disulphide groups in the granules of the secretory portion of the ducts of rat submandibular glands has been demonstrated with methods that reveal thiol groups formed after reducing the disulphide groups first. Disulphide groups were also demonstrated with cationic dyes by staining the cysteic acid residues obtained after oxidation with permanganate. Alcian Blue at pH 3.0 was used for this purpose. Two kinds of granules, characterized by their reactions with Alcian Blue at different pH levels, were apparent in differing stages of the same secretion.     

An alternative Alcian Blue dye variant for the evaluation of fetal cartilage

BACKGROUND: The most comprehensive evaluation of vertebrate skeletal development involves the use of Alizarin Red S dye to stain ossified bone and various other dyes to stain cartilage. The dye used most widely to stain fetal cartilage in rodents and rabbits is Alcian Blue 8GX. However, the global supply of this specific dye has been exhausted. Several forms of the dye marketed as Alcian Blue 8GX are now available, although they are not synthesized via the original 8GX manufacturing process. METHODS: One new Alcian Blue 8GX form and two Alcian Blue dye variants were evaluated in rats and rabbits using standard staining procedures. The staining quality of these dyes were evaluated relative to the original form of Alcian Blue 8GX based on cartilage uptake of the dye, clarity of the cartilaginous components, staining intensity of the dye, and overall readability of the specimens under stereomicroscopic evaluation. RESULTS: Staining with the newer form of Alcian Blue 8GX resulted in poor staining quality. The Alcian Blue-Pyridine variant performed well, although staining intensity was less than optimal. The Alcian Blue-Tetrakis variant provided staining characteristics that were most similar to the original form of Alcian Blue 8GX. CONCLUSIONS: Alcian Blue-Tetrakis was markedly better in its ability to stain fetal cartilage than the newer form of Alcian Blue 8GX.     

Histochemical evidence of a functional heterogeneity in neonatal canine epiphyseal chondrocytes

Synopsis The chondrocytes of the neonatal proximal humeral chondroepiphyses of twelve purebred English pointer pups were investigated histochemically, using frozen serial sections, for chondroitin sulphate and for the following enzyme activities: lactate dehydrogenase, NAD and NADP transhydrogenases, glutamate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, ATPase, succinate dehydrogenase, and UDPgalactose 4-epimerase. By using Alcian Blue with and without a prior digestion in testicular hyaluronidase, and Alcian Blue in the presence of 0.9 M magnesium chloride, it was found that about half the chondrocytes stained as if they were producing significant amounts of chondroitin sulphate. Only one enzyme, UDPgalactose 4-epimerase (which is involved in the biosynthesis of chondroitin sulphate), was found to have a similar staining heterogeneity. Therefore, it was concluded that the chondrocytes studied possessed a functional heterogenicity with particular reference to chondroitin sulphate synthesis while appearing morphologically homogeneous.     

Fixation and staining of granules in mucosal mast cells and intraepithelial lymphocytes in the rat jejunum, with special reference to the relationship between the acid glycosaminoglycans in the two cell types

Summary Various fixation and staining procedures have been examined in order to obtain optimal numbers and acceptable morphology of the mucosal mast cells and granular intraepithelial cells in the rat jejunum. For subsequent staining with Alcian Blue, the best fixation of the jejunum was obtained with a methanol-formaldehyde-acetic acid mixture. Specific staining of the granules of these cells has been obtained using Alcian Blue at pH 5.8, at which hydrogen ion concentration more cells stain than in the usual very acid conditions. Specificity is achieved by the use of magnesium chloride concentrations above the critical electrolyte concentrations for staining of protein and nucleic acid by Alcian Blue, and by the use of Safranin O as a competitive counterstain.The critical electrolyte concentration technique has also been applied to a comparative study of the glycosaminoglycan in the two cell types. Evidence is presented that the glycosaminoglycan in the granular intraepithelial cell has either a lower degree of sulphation or a lower molecular weight or both than the material in mucosal mast cells. This finding may support the possibility that the granular intraepithelial lymphocyte is a precursor of the mucosal mast cell.     

Histochemical analysis of urea-unmasked glycosaminoglycans in the skin of the rat and mouse

Summary Histochemical analysis of urea-unmasked glycosaminoglycans has been performed in connective tissues of the rat and mouse skin by means of combined staining and enzyme digestion procedures. The staining procedures used were Alcian Blue pH 1.0, Alcian Blue pH 2.5, Aldehyde Fuchsin, periodic acid-Schiff (PAS), Alcian Blue pH 2.5-PAS, high iron diamine and low iron diamine methods. The digestive enzymes employed wereStreptomyces and testicular hyaluronidases, chondroitinases ABC and AC and keratanase. The results obtained indicated that the major components of the glycosaminoglycans in the connective tissues of the skin were hyaluronic acid, dermatan sulphate and chondroitin sulphate A and/or C, whereas the tissues were devoid of keratan sulphate.     

Antifeeding activity of isoquinoline alkaloids identified in Coptis japonica roots against Hyphantria cunea (Lepidoptera: Arctiidae) and Agelastica coerulea (Coleoptera: Galerucinae)

The anti-feeding activity of 3 isoquinoline alkaloids identified from roots of Coptis japonica Makino toward 4th-instar larvae of Hyphantria cunea Drury and adults of Agelastica coerulea Baly was examined using the leaf-dipping bioassay. The biologically active constituents of the Coptis roots were characterized as the isoquinoline alkaloids berberine, palmatine and coptisine by spectroscopic analysis. In a test with H. cunea larvae, the anti-feeding activity was much more pronounced in an application of a mixture of palmatine iodide and berberine chloride (1:1, wt:wt) at 250 ppm (82.3%) and 500 ppm (100%), compared with palmatine iodide (76.0%) and berberine chloride (75.4%) alone at 500 ppm. These results indicate a synergistic effect. With A. courulea adults, berberine chloride showed 57.5 and 91.1% anti-feeding activity at 125 and 250 ppm, respectively; whereas, weak activity was obtained in application of 500 ppm of palmatine iodide (41.4%) and coptisine chloride (52.4%) alone. The Coptis root-derived compounds merit further study as potential insect-control agents.