Alcian Blue Solution Research Articles

Optimal Combination of Soluble Factors for Tissue Engineering of Permanent Cartilage from Cultured Human Chondrocytes

Since permanent cartilage has poor self-regenerative capacity, its regeneration from autologous human chondrocytes using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders. However, the conventional autologous chondrocyte implantation is insufficient both in quantity and in quality due to two major limitations: dedifferentiation during a long term culture for multiplication and hypertrophic differentiation by stimulation for the redifferentiation. To overcome the limitations, this study attempted to determine the optimal combination in primary human chondrocyte cultures under a serum-free condition, from among 12 putative chondrocyte regulators. From the exhaustive 2(12) = 4,096 combinations, 256 were selected by fractional factorial design, and bone morphogenetic protein-2 and insulin (BI) were statistically determined to be the most effective combination causing redifferentiation of the dedifferentiated cells after repeated passaging. We further found that the addition of triiodothyronine (T3) prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes via the suppression of Akt signaling. The implant formed by the human chondrocytes cultured in atelocollagen and poly(l-latic acid) scaffold under the BI + T3 stimulation consisted of sufficient hyaline cartilage with mechanical properties comparable with native cartilage after transplantation in nude mice, indicating that BI + T3 is the optimal combination to regenerate a clinically practical permanent cartilage from autologous chondrocytes.

A rapid spectrophotometric method for the determination of transparent exopolymer particles (TEP) in freshwater

A simple and rapid spectrophotometric method is proposed for the determination of transparent exopolymer particles (TEP) in freshwater samples. In this method, TEP reacts with excess of alcian blue solution yielding a low solubility dye–TEP complex. After centrifugation, the concentration of the remaining dye in the supernatant was determined at 602nm and its concentration was related to the concentration of TEP in freshwater. The effect of alcian blue concentration from 1.5×10−3 to 9.0×10−3% (m/v), solution pH from 2.5 to 6.9 and stirring time from 20 to 120s on the analytical curve was investigated. Under the optimum conditions established, such as alcian blue concentration of 3.0×10−3% (m/v); pH of 4.0 (0.2moll−1 acetate buffer solution) and stirring time of 1min, the analytical curve was linear from 0.50 to 10μgml−1 (A=0.34−0.037[GX]; r2=0.9999; where A is the absorbance and [GX] the gum xanthan concentration in μgml−1) with a detection limit of 0.10μgml−1. The recovery of TEP (as gum xanthan) for two samples ranged from 95.3 to 108 and the relative standard deviations (R.S.D.s) were lower than 0.8% for gum xanthan solutions at concentrations of 1.0 and 1.5μgml−1 (n=8). The results obtained for TEP in freshwater samples using the proposed spectrophotometric method and those obtained using a literature method are in agreement at the 95% confidence level and within an acceptable range of error.

A direct-staining method to evaluate the mucoadhesion of polymers from aqueous dispersion

A novel technique to evaluate polymer adhesion to human buccal cells following exposure to aqueous polymer dispersion, both in vitro and in vivo, is described. Adhering polymer has been visualised by staining with 0.1% (w/v) of either Alcian blue (60 min) or Eosin (10 min) solution, uncomplexed dye being removed by 0.25 M sucrose washings. The extent of polymer adhesion was quantified by measuring the relative staining intensity of control and polymer-treated cells by image analysis. In vitro, Carbopol 974P, polycarbophil (Noveon AA-1) and chitosan (CL 113) were found to adhere to human buccal cells from 0.10% (w/w) aqueous dispersions of these polymers. Following in vivo administration as a mouthwash, these polymers persisted upon the human buccal mucosa for at least 1 h.

Pleural Tissue Hyaluronan Produced by Postmortem Ventilation in Rabbits

We developed a method that used Alcian blue bound to hyaluronan to measure pleural hyaluronan in rabbits postmortem. Rabbits were killed, then ventilated with 21% O2--5% CO2--74% N2 for 3 h. The pleural liquid was removed by suction and 5 ml Alcian blue stock solution (0.33 mg/ml, 3.3 pH) was injected into each chest cavity. After 10 min, the Alcian blue solution was removed and the unbound Alcian blue solution (supernatant) separated by centrifugation and filtration. The supernatant transmissibility (T) was measured spectrophotometrically at 613 nm. Supernatant Alcian blue concentration (Cab) was obtained from a calibration curve of T versus dilutions of stock solution Cab. Alcian blue bound to pleural tissue hyaluronan was obtained by subtracting supernatant Cab from stock solution Cab. Pleural tissue hyaluronan was obtained from a calibration curve of hyaluronan versus Alcian blue bound to hyaluronan. Compared with control rabbits, pleural tissue hyaluronan (0.21 +/- 0.04 mg/kg) increased twofold, whereas pleural liquid volume decreased by 30% after 3 h of ventilation. Pleural effusions present 3 h postmortem without ventilation did not change pleural tissue hyaluronan from control values. Thus ventilation-induced pleural liquid shear stress, not increased filtration, was the stimulus for the increased hyaluronan produced from pleural mesothelial cells.

Use and Advantages of Ethanol Solution of Alizarin Red S Dye for Staining Bone in Fishes

A solution of 75% ethyl alcohol (ETOH) and alizarin red S can be substituted for KOH-alizarin red S to stain bone. ETOH-alizarin, unlike KOH-alizarin, does not damage muscle or cartilage. ETOH-alizarin and acidified alcian blue (a cartilage stain) can be used in any order and as often as required to stain bone and cartilage in a single preparation that has not been exposed to KOH-alizarin or trypsin, remembering that the acidic effects of the alcian blue solution should be neutralized to prevent decalcification of weakly ossified elements.

Study on the experimental ulcerative colitis model induced by dextran sulfate sodium in rats: estimation of mucosal erosions by the alcian blue-staining method

We have developed a new method that is applicable for a macroscopic and objective evaluation of erosion in the large intestine in the experimental ulcerative colitis model induced by dextran sulfate sodium (DSS) in rats. The large intestine (without cecum) fixed in 10% neutral buffered formalin solution for more than one week was opened and stained with 1% alcian blue solution. The mucosal surface was stained in light and shade-blue. Histopathological examination revealed that the dark blue area on the mucosal surface had no epithelia and that the connective tissues in the lamina propria were stained with alcian blue. Salazosulfapyridine at 15 and 50 mg/kg twice a day inhibited the erosion area (dark blue area) by 29.6% and 50.2%, respectively. Also, prednisolone at 0.5 mg/kg, twice a day inhibited the erosion by 53.3%. Thus, by measuring the dark blue area stained with 1% alcian blue solution, we could estimate macroscopically and objectively the area of erosions. This method seems to be a useful index for assessing the damages produced in the experimental ulcerative colitis model in rats.

Optimization of an alcian blue dot-blot assay for the detection of glycosaminoglycans and proteoglycans

A modification of a method for the detection of proteoglycans on positively charged nylon was adapted for use in a dot-blot assay and improved. Different dot-blot membranes were tested and variations in the Alcian blue staining solution (including pH, critical electrolyte concentration, and ionic strength) were explored. With modifications, we were able to eliminate interferences by other polyanions such as DNA or proteins. We were able to detect glycosaminoglycans and proteoglycans down to the 10 ng range. Furthermore, our assay is compatible with high concentration of urea (up to 7 m) used in classic proteoglycans extraction methods and is a useful tool to monitor the isolation of proteoglycans by anion exchange and gel filtration chromatography. It is technically easier, faster, more sensitive, and more specific than previously published methods and can be adaptated as a quantitative assay using a scanning densitometer with a linear range of detection from 10 to 100 ng of glycosaminoglycans.

Micro-PIXE quantification of histochemical reactions

The proton induced X-ray emission in combination with a proton microprobe (micro-PIXE) was used for the quantitative determination of a locally bound histochemical reaction end-product. The determination of proteoglycans (PG) content in the cartilage is described in detail. The cartilage from the femur and the skull of 17–19 days old embryos and from ribs of mature CBA strain mice was investigated. The 8 μm cryomicrotome sections, placed on a formvar backing, were incubated in colloidal iron or in alcian blue (dye molecule contains Cu) solutions. The determination of PG contents was made indirectly by assessment of the Fe or Cu concentrations. Differences in the PG contents were observed within all investigated cartilage types. The embryonal cartilage contains more PG than that obtained from mature animals. For the cartilage from the femur the highest content of PG was found in the mineralization front while fully formed bone trabeculae exhibited a PG level 10 times lower. In the case of intramembranaceous ossification the content of PG was also higher in the skull cartilage than in the loose connective tissue. On the basis of the performed studies some methodological conclusions can be drawn. For PG determination, the alcian blue method is more reliable than the colloidal iron technique. The review of the histochemical procedures which may be combined with the micro-PIXE method is also given.

Alcian Blue staining of cochlear hair cell stereocilia and other cochlear tissues

Using whole-mount surface preparations of the chinchilla organ of Corti, the anionic characteristics of cochlear tissues was investigated with the polycationic dye Alcian Blue. Our primary goal was to specifically stain the hair cell stereocilia for the purpose of producing cytocochleograms. To specifically stain hair cell stereocilia we investigated the effects of different fixatives and the 'critical electrolyte concentration' [Scott and Dorling (1965) Histochemie 5, 221-233] of MgCl2 on staining specificity. The stereocilia and the tectorial membrane were the most intensely stained cochlear structures and presumably have a strong negative charge. The most important factor in producing specific staining was fixation in osmium tetroxide rather than the addition of an electrolyte to the Alcian Blue staining solution. The use of Alcian Blue produced intense staining of hair cell stereocilia against an unstained background using standard, brightfield light microscopy.

Histochemical demonstration of cytoplasmic glycosaminoglycans in the macroneurons of the human central nervous system.

The presence of glycosaminoglycans (GAG) has been histochemically demonstrated in the CNS of various mammalian species. They have been related with some nerve functions as neurotransmitters storage and synaptic transmission. In the present paper, the histochemical properties of nerve cell cytoplasmic GAG were studied in several regions of adult human CNS. Samples of brain cortex, pons, upper medulla, and cerebellar cortex obtained by autopsy from subjects not dying after neurological diseases were fixed by immersion in glutaraldehyde, dehydrated with ethanol, and embedded in paraffin. The sections were stained with Alcian blue solutions adjusted to pH 2.5, 4.0, and 5.7. To the latter solution MgCl2 was added in increasing concentration from 0.05 to 1.2 M. Testicular hyaluronidase, neuraminidase, and ribonuclease were applied on simultaneous sections with their respective controls. The sequence of these reactions allowed us to demonstrate the presence of hyaluronic acid along chondroitin-4- and/or 6-sulphate in the cytoplasm of most nerve cells. The sulphated GAG showed certain variability in the various regions studied related specially with their grade of sulphation.

Cortical granule exocytosis and cell surface reorganization in eggs of Brachydanio.

AbstractCortical granules in the mature, unfertilized egg of the zebra danio (Brachydanio rerio) are spherical, membrane‐bound organelles measuring 3 to 22 μm in diameter and arranged in irregular rows beneath the plasma membrane. Two types of cortical granules are identified in thin sections viewed under the transmission electron microscope: “dark” type (electron‐dense core) and “light” type (electron‐lucent core). Cytochemically, glycoproteins and acid mucopolysaccharides are constituents of the cortical granules. Differences in staining reactions with Alcian blue and toluidine blue solutions also reveal the presence of two types of cortical granules in the danio egg. The “dark” type of cortical granule stains strongly with both dyes and shows a single, central core surrounded by a zone of lighter, particulate material. The “light” type of cortical granule stains faintly with both dyes. Differences in staining responses are not related to the size of the cortical granules.Analysis of the cortical granule reaction in inseminated eggs by light microscopy reveals that breakdown and discharge of cortical granules begin after 30 seconds postactivation. Since the chorion is lifted away from the egg surface by this time, the initial elevation of this membrane occurs independently of the onset of cortical granule exocytosis. The breakdown and discharge of cortical granules is a prominent feature of egg surface activity between 1 and 3 minutes postactivation. The cortical granule reaction is completed by 5 to 6 minutes postactivation.Scanning electron microscopy has provided an unusual opportunity to observe in detail cortical granule exocytosis and the reorganization of the egg surface during the cortical reaction. Cortical granule exocytosis consists of the following sequence of morphological stages: elevation of the cortical granule above the egg surface, rupture of the membrane covering the cortical granule, and discharge of central and peripheral constituents of the cortical granule into the perivitelline space. Rupture of the cortical granule follows contact and fusion between perigranular and egg plasma membranes and is effected through the coalescence of fissures that appear in the egg surface between the equator and the top of the elevated granule.Our SEM and TEM findings suggest strongly that the mosaic surface formed upon cortical granule exocytosis is temporary and that perigranular membranes have limited residence in the egg surface. Morphological evidence presented here is consistent with the view that the zebra danio egg accommodates the excessive membrane introduced into its surface during the cortical reaction by endocytosis. It is proposed that perigranular membrane is retrieved continuously after exocytosis but is visible morphologically as a two‐step process. First, perigranular membrane is removed from the egg surface into the cortical cytoplasm in the form of vesicles (0.5–1.5 μm in diameter) pinched‐off from the base of the cortical crypt. Second, localized folds of the egg plasmalemma overgrow and replace, perhaps through contractile activity, remaining perigranular membrane at the egg surface.

Cutaneous Calcinosis in Localized Discoid Lupus Erythematosus

Cutaneous calcinosis is reported in the lesions of a woman with long-standing discoid lupus erythematosus. Calcium deposits were found in the middle and deep parts of the dermis surrounded by homogeneous collagen staining positively with Alcian blue solution. In contrast to the relative frequency of this event in patients with scleroderma or dermatomyositis, calcinosis cutis occurs very rarely in lupus erythematosus. To the best of our knowledge, this is the first report of calcinosis cutis in discoid lupus erythematosus.

Cutaneous calcinosis in localized discoid lupus erythematosus

• Cutaneous calcinosis is reported in the lesions of a woman with long-standing discoid lupus erythematosus. Calcium deposits were found in the middle and deep parts of the dermis surrounded by homogeneous collagen staining positively with Alcian blue solution. In contrast to the relative frequency of this event in patients with scleroderma or dermatomyositis, calcinosis cutis occurs very rarely in lupus erythematosus. To the best of our knowledge, this is the first report of calcinosis cutis in discoid lupus erythematosus. (<i>Arch Dermatol</i>116:196-197, 1980)

A colorimetric assay for mucous glycoproteins using Alcian Blue [proceedings

The present paper describes a simple, sensitive technique for measurement of respiratory mucous glycoproteins that involves their precipitation with the cationic dye Alcian Blue. The assay was adapted from that described by Whiteman (1973) for measurement of urinary glycosaminoglycans. This method is useful for measurement of mucins secreted in response to stimulating agents in isolated airway preparations of animals, and of human airway secretions obtained by bronchoswpy. The procedure is also useful for measurement of mucin content of fractions obtained after column chromatography. The Alcian Blue assay was developed as an alternative to the usual methods of glycoprotein determination, which measure total hexose wntent (phenol/sulphuric acid, anthrone, orcinol), which are of limited sensitivity (Mantle & Allen, 1978) and are hazardous to operate, requiring the use of large amounts of sulphuric acid. Mucous glycoproteins are polyanionic, having both sialic acid and sulphate residues attached to their oligosaccharide structures. It is these groups that interact with Alcian Blue to form insoluble complexes. A 0.1% (w/v) solution of Alcian Blue in 0.1 M-sodium acetate/acetic acid buffer, pH 5.8, containing 25 mM-MgC1, was clarified by sedimentation at 1870ga, for 30min at 2OoC in an MSE Mistral 2L centrifuge. To 3.0ml portions of tracheal mucus samples in plastic test tubes was added 1 ml of the purified Alcian Blue solution. After at least 2 h equilibration at room temperature, mucus-Alcian Blue complexes were sedimented at 1870ga, for 30min at 2OOC. Supernatant solutions were discarded, and each pellet was washed twice by successive resuspension in 40% (v/v) ethanoV0.1 Msodium acetate buffer, pH 5.8, containing 25 mM-MgCI,, and sedimentation at 1870ga, for IOmin at 2OOC. A 40% (w/v) Manoxol 1 B (sodium dibutylsulphosuccinate) solution was clarified by filtration. Mucus-dye complexes were dissociated by addition of 1 ml of the Manoxol 1B solution and ultrasonication at SOW for 10s with a Dawes soniprobe. Samples were sedimented for 1 min as described above to eliminate the foam generated during sonication. Absorbance at 620nm was measured in a Cecil CE272 spectrophotometer. A calibration curve for Alcian Blue precipitation was determined for a cat tracheal mucus preparation (Fig. 1) obtained as described by Gallagher et al. (1975). The protein content of this preparation was determined by alkaline hydrolysis followed by ninhydrin reaction, and the carbohydrate content was determined by g.1.c. (Hall, 1978). The carbohydrates present were those typical of cat tracheal mucins (Gallagher et al., 1975) and contained no deoxyribose, ribose or uronic acids. This mucus preparation was not contaminated by nucleic acids or proteoglycans. The resultant absorbance at 620nm of the Alcian Blue 0.6 r

Histochemistry of the intercellular substance of the normal and psoriatic human epidermis.

The intercellular substance of skin samples obtained from normal subjects and from psoriatic patients has been studied with histochemical methods for carbohydrate containing substances and checked with enzymatic extractions. The surface coat which makes up most of the intercellular substance was stained with colloidal iron and with Alcian Blue solutions containing up to 0.20 M magnesium chloride; the stainings were heavily affected by the previous treatment of the sections with testicular hyaluronidase, but not with neuraminidase. The staining of the intercellular substance with Alcian Blue solutions containing up to 0.20 M magnesium chloride and the action of the hyaluronidase gives strength to the hypothesis that hyaluronic acid is contained in the substance. In the skin of psoriatic patients intercellular spaces wider than in normal skin and a reduced surface coat, particularly in the higher layers, has been observed.

Electron microscopic demonstration of hyaluronidase sensible proteoglycans at the presynaptic area in mouse cerebellar cortex

By application of electron cytochemical techniques to cerebellar tissue, the presence of proteoglycans was demonstrated at the axoplasmic matrix of mossy fiber endings. Blocks of glutaraldehyde (G) fixed mouse cerebellum were processed according to the following procedures: a) Some pieces of tissue were post-fixed in osmium tetroxide, dehydrated by ethanol and embedded in araldite. b) Other pieces were sectioned to 30 mum thick and then immersed in Alcian blue solution pH = 2.7 followed by osmium tetroxide fixation, dehydrated and embedded in araldite (GABOUL procedure). c) Parallel slices of (b) previous to Alcian blue immersion were washed and incubated in either methanol-HCl, neuraminidase, ribonuclease or testicular hyaluronidase with their respective controls. d) Other blocks of G fixed tissue without any other treatment and fixation were dehydrated and embedded in araldite. Ultrathin sections of a, b and c were doubly stained with uranyl acetate and lead citrate while ultrathin sections of (d) were stained with the osmium coordination compound Os-DMEDA. The electron microscopic study revealed at the presynaptic axoplasm of mossy fiber rosettes, the presence of a GABOUL and Os-DMEDA positive electron dense material surrounding synaptic vesicles and continuous with presynaptic dense projections. This material which coincides with cytonet distribution was resistant to neuraminidase and ribonuclease and sensible to hyaluronidase and carboxymethylation. These findings permit us to conclude that the axoplasmic material of mossy fiber endings is constituted by proteoglycans in which hyaluronic acid and chondroitin 4-and/or 6-sulphate are present. The probable importance of these proteoglycans in synaptic mechanisms is also discussed.

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.

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.

Microanalysis and characterization of acidic glycosaminoglycans in human tissues

The acid glycosaminoglycans (AG) were isolated from small quantities of human tissue (1.0 gm wet weight or less) and were separated, identified, and measured by zone electrophoresis on cellulose acetate. The major AG components in human skin, sclera, and cornea were identified and measured. The amount of Alcian Blue (dye used to stain the AG) bound to each AG polymer on the cellulose acetate strip was dependent on the number of moles of disaccharide repeating units (DRU) times the number of dissociated carboxylic and sulfate groups per DRU. The degree of dissociation of these groups depended on their corresponding p K's and the pH of the Alcian Blue solution. The glucosamine/galactosamine fraction agreed closely with the hyaluronic acid/sulfated AG (dermatan sulfate + chondroitin 4 6 - sulfate ) fraction obtained by electrophoresis. The mobility of each AG polymer was affected more by the number of negative charges per DRU than by the molecular weight. The identity and number of charged groups per DRU could be deduced from the electrophoretic mobilities and the dye binding data. The combined use of chemical analyses and zone electrophoresis permitted the major AG polymers to be identified and measured in tissues without the use of ion-exchange columns.

Effect of Fixatives on Acid Mucopolysaccharides (AMPS) in the Experimentally Hydrated Rabbit Cornea

The influence of various fixatives on the rabbit cornea hydrated in distilled water for 2 and 4 h was studied by means of staining with Alcian stains at various pH, colloidal Fe³⁺, Alcian blue solutions with increasing concentrations of MgCl₂, and toluidine blue at pH 4.5. The initial hydration resulted in an increased intensity of staining of carboxyl groups and with the stainability of sulphate groups. These changes induced by primary hydration remained qualitatively unchanged after treatment with different fixatives. Differences were observed only in degrees of staining intensities. In comparison with other fixatives, formol-cetylpyridinium chloride (CPC) fixation significantly decreased the staining intensity of stromal AMPS.

Histochemistry of Mucosubstances in the Mantle of the Clam, Mercenaria mercenaria. II. MucoSubstances in the Second Marginal Fold

Histochemistry of Mucosubstances in the Mantle of the Clam, Mercenaria mercenaria. II. MucoSubstances in the Second Marginal Fold