alcian-blue and copper-phthalocyanine

A brief outline of the history and chemistry of the copper pthalocyanin-based Alcian blue dyes highlights the adverse effects of commercial secrecy and entrepreneurial dishonesty on the use of these valuable histochemical and biochemical reagents in the biological sciences. The acquisition of detailed and validated chemical structures required the avoidable expenditure of much time that otherwise would have been spent on new research. The combination of this chemical knowledge with experiments on isolated polyanions, using critical electrolyte concentration (CEC) techniques, established a molecular recognition paradigm of Alcian blue usage in histochemistry. New reagents (Cuprolinic and Cupromeronic blues) were synthesised to investigate and support the hypothesis that Alcian blue did not intercalate with DNA. They were made with the important additional aim of using them in electron microscopy of polynucleotides (in the case of Cuprolinic blue) and non-nucleotide polyanions, such as sulphated proteoglycans in extracellular matrices (in the case of Cupromeronic blue). The cationic groups of Alcian blue are easily removed in very mild conditions, producing insoluble blue pigment in situ (ingrain dying). This reaction underlies its unique capability to stain in repeated cycles, thus greatly amplifying sensitivity of detection of substrates present in very small amounts in tissues and on electrophoretic strips etc.

Topics: Alcian Blue; Animals; Chemical Industry; Coloring Agents; Electrolytes; Extracellular Matrix; Glycosaminoglycans; Histocytochemistry; History, 20th Century; Humans; Indicators and Reagents; Indoles; Molecular Structure; Organometallic Compounds; Structure-Activity Relationship

Alcian blue dyes are copper phthalocyanines with a variety of cationic side chains; they are useful for staining carbohydrate polyanions while avoiding staining of nucleic acids. The properties of the original alcian blue and of similar dyes with published chemical structures are reviewed here. Variation among samples submitted to the Biological Stain Commission (BSC) for certification has led to the recognition of two types of commercially available alcian blue at this time. The designation "alcian blue 8G or equivalent" is reserved for dyes that resemble alcian blue 8GX manufactured in the 1960s (CI 74240; ingrain blue 1). These dyes react with alkali to form an insoluble pigment that cannot be re-dissolved in acid. The name "alcian blue variant" is for similar dyes that do not form insoluble pigments; an alkali-induced precipitate, if formed, re-dissolves with acidification. For certification by the BSC, both types of alcian blue must dissolve in 3% acetic acid to make a 1% solution (pH close to 2.5), which must provide selective coloration of intestinal mucus, cartilage and mast cells, but not of nuclei. After alcian blue staining and treatment with 0.03 M Na

Topics: Alcian Blue; Animals; Cartilage; Certification; Coloring Agents; Hydrogen-Ion Concentration; Indoles; Organometallic Compounds; Reference Standards; Staining and Labeling

We have found that a positively charged cationic copper phthalocyanine, Alcian blue (Alcian blue 8GX), can efficiently quench the fluorescence of an oppositely charged red fluorescent phthalocyanine compound with a matched molecular structure, tetrasulfonated aluminum phthalocyanine (AlS4Pc), because of the formation of an ion pair complex (AlS4Pc-Alcian blue 8GX) that exhibits almost no fluorescence. An investigation was carried out on the fluorescence recovery of AlS4Pc-Alcian blue 8GX caused by a series of anionic surfactants containing a sulfonic group (sodium dodecylbenzenesulfonate (SDBS), sodium lauryl sulfate (SLS), and sodium dodecyl sulfate (SDS)). The results showed that SDBS exhibited a significant response, and the highest sensitivity among the surfactants. Due to its high efficiency of fluorescence quenching and the high level of fluorescence recovery, direct observes can even be performed by the naked eye. The results revealed that the Alcian blue 8GX-AlS4Pc ion-pair complex fluorescent probe only responded to SDBS in the low-concentration range. Based on the new founding, this study proposed a novel principle and method of fluorescence enhancement to specifically measure the concentration of SDBS, thereby achieving a highly sensitive and highly specific determination of SDBS. Under the optimal conditions, the fluorescence intensity (I(f)) of the system and the concentration of SDBS in the range of 1 × 10(-7) - 1 × 10(-5) mol/dm(3) exhibited a good linear relationship. This method is highly sensitive, and the operation is simple and rapid. It had been applied for the quantitative analysis of SDBS in environmental water, while achieving satisfactory results compared with those of the standard method. This study developed a new application of the fluorescent phthalocyanine compounds used as molecular probes in analytical sciences.

Topics: Alcian Blue; Anions; Benzenesulfonates; Copper; Fluorescent Dyes; Indoles; Isoindoles; Molecular Structure; Optics and Photonics; Organometallic Compounds; Sodium Dodecyl Sulfate; Solubility; Spectrometry, Fluorescence; Surface-Active Agents

Ultrastucture of the tectorial membrane in the chinchilla cochlea was studied by transmission electron microscopy using different fixatives and staining procedures. It was shown that the tectorial membrane is a highly structured matrix composed of collagen type A fibrils, noncollagenous type B fibrils and proteoglycan. The localization of type B fibrils surrounding bundles of parallel type A fibrils was observed. Staining of the tectorial membranes with the cationic dye Cuprolinic blue in a "critical electrolyte concentration" method revealed proteoglycan, D-periodically associated with collagen type A fibrils and orthogonal to them. The appearance and size of the proteoglycan, and its binding to collagen, were similar to small proteoglycans observed in cartilage and other tissues. In many regions of the tectorial membrane the collagen-bound proteoglycan forms crystalline-like arrays. The images of these arrays processed by Fourier analysis show long linear aggregates of proteoglycan arranged parallel each other.

Topics: Alcian Blue; Animals; Chinchilla; Collagen; Coloring Agents; Indoles; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Ruthenium Red; Tectorial Membrane

To examine the distribution of proteoglycans in the exfoliation materials in order to investigate the nature of the materials.. The anterior parts of two eyes with exfoliation syndrome were examined by electron microscopy after staining with cupromeronic blue (cmb). Some specimens were treated with enzymes and/or nitrous acid prior to staining. The effects of the enzymes were evaluated statistically by counting the density of the cmb-positive filaments in the exfoliation materials, using a computer. One eye with exfoliation syndrome stained with alcian blue was observed with light microscopy.. Exfoliation materials were observed along the epithelial cells of the iris and ciliary body, and in the trabecular meshwork and zonules. In tissue specimens treated with cmb, electron-dense filaments were seen associated with the exfoliation materials. Microfibrils in the trabecular meshwork and iris, and zonular fibrils themselves were free of any filament staining, while the exfoliation materials located closely to the fibrils contained the electron-dense filaments. In the tissue specimens treated with chondroitinase AC, chondroitinase B, chondroitinase ABC or nitrous acid before cmb staining, the amount of the filament associated with exfoliation materials decreased in comparison to the controls. Digestion with keratinase did not demonstrate any significant changes in staining. A combination treatment with chondroitinase ABC and nitrous acid eliminated almost all filaments associated with the exfoliation materials. In the eye stained with alcian blue, the zonules that did not stain for the dye demonstrated an accumulation of exfoliation materials that stained strongly for alcian blue.. Exfoliation materials contain chondroitin sulfate, dermatan sulfate, heparan sulfate proteoglycans. Depositions of proteoglycans on the microfibrils may be closely associated with the formation of exfoliation materials.

Topics: Aged; Aged, 80 and over; Alcian Blue; beta-Galactosidase; Chondroitin Lyases; Ciliary Body; Coloring Agents; Exfoliation Syndrome; Female; Glycoside Hydrolases; Humans; Indoles; Iris; Male; Nitrous Acid; Organometallic Compounds; Proteoglycans; Trabecular Meshwork

The time course of development of extracellular matrix (ECM) in experimentally induced fibrosis (thioacetamide administration followed for 12 weeks or bile duct ligation for 8 weeks) in adult rats was examined by light and electron microscopy, using Alcian blue or Cupromeronic blue staining for sulphated proteoglycans (PGs) in critical electrolyte concentration techniques. Proteodermatan sulphate (PDS) was regularly observed at the gap zone of the collagen fibrils. Morphometry of uranyl acetate-stained collagen fibrils, polarity of their banding patterns (a-e), statistics of d/e band occupancies by PDS, and lengths and thicknesses of PG filaments were quantified. Biochemical analyses showed that the ECM components collagen, hyaluronan, chondroitin and dermatan sulphates increased by 5-10 fold, roughly in parallel, as did heparan sulphate and DNA. Water and lipid contents also increased sharply. Thioacetamide treatment was much slower than bile duct ligation in producing fibrotic changes of equal severity. Sulphation of anionic glycosaminoglycans (AGAGs) decreased with increasing severity of fibrosis. Biochemical and ultrastructural methods correlated well. The large increase in dermatan sulphate was quantitatively as expected, given that it is collagen fibril surface-associated, and there was an increase of collagen content together with a decrease in fibril thicknesses. The increase in DNA reflected the marked increase in cell numbers in fibrotic livers. The chemical morphology of the new connective tissue closely resembled that in e.g. developing young tendon, in that fibrils were thinner, and AGAG levels were higher. The collagen fibrils were often disarranged, rather than ordered and parallel as in normal ECM. No other indication of abnormality in the new ECM was obtained.

Topics: Alcian Blue; Animals; Chondroitin Sulfates; Collagen; Connective Tissue; Dermatan Sulfate; DNA; Extracellular Matrix; Female; Hyaluronic Acid; Indoles; Liver Cirrhosis, Experimental; Male; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Rats; Rats, Sprague-Dawley; Staining and Labeling

Proteoglycans (PGs) in the epithelial-stromal interface of the guinea pig lateral prostate were localized at ultrastructural level, using cuprolinic Blue (CB), alcian Blue (AB), and ruthenium red (RR). After staining with CB or AB according to the critical electrolyte concentration method (CEC), PGs appeared as short electron-dense filaments. According to their sizes and location, three type (T1, T2, T3) of CB-stained filaments were identified. T1 filaments were short (25 nm) and were found on both sides of the lamina densa of the basal laminae of the prostatic epithelium, smooth muscle cells, and capillary endothelial cells. They were regularly spaced with an interval of 60 nm. T1 filaments were more randomly distributed in the lamina densa. T2 CB filaments were approximately 30-40 nm long and closely associated with the collagen fibrils. They were usually arranged perpendicular to the long axis of collagen fibrils also at intervals of 60 nm. T3 filaments were found in different regions of the lamina propria, including: 1) reticular layer (pars fibroreticularis) below the basal lamina; 2) interstitial spaces; 3) closely associated with the cell surfaces of fibroblasts; and 4) around the collagen fibrils. Their sizes were variable (60-100 nm) and more densely stained. AB revealed similar patterns of PG distribution, except that the three types of PG filaments were longer but thinner. When the tissues were stained with RR, or RR-AB combined, PGs appeared as dense granules of various sizes, instead of filaments. Their locations and distributions were similar to those of the CB filaments, except that in the case of combined RR-AB treatment the PG granules were linked by a fine filamentous network, suggesting the interconnecting nature of the PGs and other extracellular components.

Topics: Alcian Blue; Animals; Cations; Coloring Agents; Epithelium; Guinea Pigs; Indoles; Male; Organometallic Compounds; Prostate; Proteoglycans; Ruthenium Red

The described method provides a new technique for differentiating areas of gray and white matter in fixed human brain slices. The technique is a modification of an existing method permitting use of the nonfading copper phthalocyanine dye alcian blue. Stained slices show turquoise gray matter that contrasts sharply with areas of white matter.. Cut human brains from gross anatomy laboratory cadavers into 4 mm slices and wash in running tap water for 12 hr. Oxidize slices in performic acid for 1.5 hr. Wash in running tap water for 12 hr. Stain slices in shallow dishes in 0.05% aqueous alcian blue. Wash in running tap water for 1 hr. Dry for 2-4 hr and embed in plastic.

Topics: Alcian Blue; Brain; Histological Techniques; Humans; Indoles; Organometallic Compounds; Staining and Labeling

In order to contrast anionic sites in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules. After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl2 concentration used during staining. At 0.4 M MgCl2, the length was mostly within the range 100-180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.

Topics: Alcian Blue; Animals; Anions; Basement Membrane; Indoles; Mice; Mice, Inbred Strains; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Pulmonary Alveoli; Ruthenium Red; Staining and Labeling

The staining of chicken erythrocyte nuclei with Alcian blue followed by a treatment with ethanol-NaOH, showed to be highly resistant to acid extraction (5 N HCl) due to the alkaline conversion of Alcian blue into the insoluble pigment Monastral fast blue. It is assumed that the intercalative binding of the planar Monastral fast blue molecule to DNA accounts for its competition effect toward the intercalating fluorochromes acridine organe, ethidium bromine and daunomycin.

Topics: Alcian Blue; Animals; Cell Nucleus; Chickens; Chromatin; Erythrocytes; Indoles; Organometallic Compounds; Staining and Labeling

Four cationic copper phthalocyanin dyes are compared with Alcian Blue 8G for specificity of interaction with a range of natural and synthetic polyanions. The CEC system is used to estimate dye-substrate affinities. The results support the hypothesis that the tissue staining pattern of Alcian Blue is determined by (a) exclusion of the dye from polynucleotide stacked base pairs by steric hindrance due to bulky substituents on the chromophore and (b) the presence of pendant quaternary charges whose affinity for negative sites on polyanionic substrates is without regard for the precise chemical nature of the anion. These conclusions, and practical considerations lead to the choice of quaternized Astra Blue 6GLL as a replacement for Alcian Blue (now no longer manufactured). It is superior to Alcian Blue in pH range, and stability in solution. It is easily prepared from a readily available precursor.

Topics: Alcian Blue; Anions; Cations; Chemical Phenomena; Chemistry; Coloring Agents; Histocytochemistry; Indoles; Organometallic Compounds; Staining and Labeling