Critical Electrolyte Concentration Method Research Articles

Myxoid tumours of soft tissue: the so‐called myxoid extracellular matrix is heterogeneous in composition

Myxoid tumours of soft tissue are characterized by their so-called 'myxoid' extracellular matrix. The aim was to investigate the composition and possible function of this matrix which is poorly understood. Using Alcian Blue staining with and without pretreatment with hyaluronidase and application of the critical electrolyte concentration method followed by densitometry, the glycosaminoglycan composition of three different myxoid tumours was studied. The composition of glycosaminoglycans varied with tumour type and grade, despite their general characterization as myxoid tumours. Intramuscular myxoma contained similar amounts of the various glycosaminoglycans as grade I myxofibrosarcoma; grade III myxofibrosarcoma contained less hyaluronic acid and more heparan sulphate, whereas extraskeletal myxoid chondrosarcoma contained predominantly chondroitin-4 and -6 sulphates. Western blot identified albumin as a major protein in tumour lysates, and its presence in the extracellular matrix and cytoplasm of the majority of tumours was demonstrated by immunohistochemistry; production of albumin by the tumour cells was confirmed by quantitative polymerase chain reaction. The extracellular matrix of myxoid tumours of soft tissue has a heterogeneous composition consisting of, amongst others, glycosaminoglycans and albumin, which appear to play an active role in their morphogenesis.

Proteoglycan distribution pattern during aging in the nematode Caenorhabditis elegans: an ultrastructural histochemical study.

Glycosaminoglycans are important constituents of the extracellular matrix of vertebrates, where distinct changes in their distribution pattern occur during aging. However, little is known about their changes in the nematode Caenorhabditis elegans, which ages extremely rapidly compared to mammals. The presence of glycosaminoglycans was analysed in cross-sections of all organs of the nematode, in three different age groups (60, 144, 228 h), using the electron-dense dye Cuprolinic Blue in conjunction with the critical electrolyte concentration method and specific glycosaminoglycan degrading enzymes. The nematodes (strain DH 26) were grown at 25.5 degrees C. The results indicate the presence of an organ-specific distribution pattern. Chondroitin-4-sulphate and/or chondroitin-6-sulphate are present in the epicuticula. Chondroitin-4-sulphate and/or chondroitin-6-sulphate and dermatan sulphate are detected in the mesocuticula. If stained by conventional methods the mesocuticula shows an empty fissure, which is filled by chondroitin sulphates and dermatan sulphate as shown by Cuprolinic Blue staining and enzymes. Heparan sulphate is found in the terminal web of intestinal cells while dermatan sulphate is revealed in the central cores of microvilli. An unknown polyanion staining at high electrolyte concentrations is observed in the gonads. Age-related changes do not impair the composition of the glycosaminoglycan fraction. In conclusion an unexpected highly differentiated pattern of glycosaminoglycans with high stability during aging exists.

Apoptosis: identification by a critical electrolyte concentration method

A critical electrolyte concentration method previously proposed to distinguish RNA from DNA was considered for a rapid screening of apoptosis at the cellular level. For the monitoring of the apoptotic phenomenon, preparations subjected to anin situ 3′ end DNA labelling assay were also analyzed. The results indicated that the critical electrolyte concentration assay as used here is a simple and useful tool for the rapid identification of apoptotic cells and could contribute to the determination of their frequency, localization and other characteristics.

Application of selected cationic dyes for the semiquantitative estimation of glycosaminoglycans in histological sections of articular cartilage by microspectrophotometry.

Selected commonly used cationic dyes, viz. Thionin, Safranin O, Toluidine Blue O, Dimethylmethylene Blue, Cuprolinic Blue, Cupromeronic Blue, N,N'-Diethylpseudoisocyanine, and a modified PAS-method, and staining methods with a variety of alternative procedures, e.g., variation of pH, use of the critical electrolyte concentration method, and blocking reactions (methylation-saponification, carboxymethylation), were tested to select optimal staining procedures for the semiquantitative histochemical estimation of glycosaminoglycans by microspectrophotometry in sections of articular cartilage. The methods were carried out on 3 microns-thick paraffin and 1 microns-thick glycolmethacrylate sections of bovine articular cartilage. The staining intensity of the sections was measured from spots 25 microns apart using a Leitz MPV 3 microspectrophotometer, starting at the surface of the cartilage and ending up at the tidemark. The result was compared with the fixed-charge density graph determined from the adjacent articular cartilage. Of the dyes tested, Thionin and Safranin O proved to be excellent cationic dyes for the histochemical quantification of cartilage matrix proteoglycans, since the staining intensity curves showed a linear correlation (r = 0.900-0.995) with the fixed charge density curves from the adjacent cartilage. Also, the stain distribution was consistently uniform across the sections. In 1 microns-thick glycolmethacrylate sections, the Safranin O staining gradient showed almost perfect identity with the fixed-charge density curve. Cuprolinic Blue and Cupromeronic Blue combined with the critical electrolyte concentration technique were also useful for the microspectrophotometric assays of glycosaminoglycans, but the presence of metachromasia should be checked prior to the measurements. The reliability of blocking procedures for quantitative histochemical work was not convincing.

Relocation of RNA Metachromasy at Mitosis.

Changes in patterns of metachromatic staining due to RNA were followed by a critical electrolyte concentration method in culture cells during mitosis, and compared with AgNOR staining results and with published data especially referring to nucleolar/nuclear proteins. A deep metachromatic response was found in association with the chromosomes, radiating along the spindle structure and then becoming greatly reduced in nuclei at late telophase/early cytokinesis. This response distribution was not exactly coincident with the reported relocation of nucleolar/nuclear proteins or with a non-nucleolar RNA-containing layer during mitosis. The sites of RNA metachromasy also differed from those of Ag-NOR positive response. The results indicate that RNA most probably of nucleolar origin participates in the complex architecture which is assumed to protect the chromosomes, to store rRNA maturation factors, and to contribute to distribution of nucleolar/nuclear proteins and particular RNA types to daughter cells.

The proteoglycan skeleton of the Kurloff body as evidenced by cuprolinic blue staining.

This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosome organelle, metachromatic towards Toluidine Blue, of a blood cell unique to the guinea pig and called the Kurloff cell. Splenic Kurloff cell from oestrogen-treated guinea pig cells were examined after staining with Cuprolinic Blue, a cationic phthalocyanine-like dye, in the presence of MgCl2 in a critical electrolyte concentration method. Better results were obtained when the fixation-staining by the glutaraldehyde Cuprinolinic Blue MgCl2 mixture was preceded by a glutaraldehyde pre-fixation. On light microscopy, Kurloff bodies generally exhibited an overall pink and glassy metachromasia, sometimes with additional darker metachromatic small dots at their peripheries. At the ultrastructural level, the metachromatic central matrix of the Kurloff body usually exhibited, as a major feature, a typical network pattern of ribbon-like or stellate electron-dense precipitates suggesting the presence of a skeleton of Cuprolinic Blue-reactive filamentous structures. Taking into account their high anionicity (as shown by the stability of the dye binding in the presence of 0.3 M MgCl2) and their susceptibility to chondroitinase ABC, these anionic structures were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell.

Application of cationic probes for the ultrastructural localization of proteoglycans in basement membranes.

The application of cationic probes for the ultrastructural detection of proteoglycans in basement membranes is reviewed. Proteoglycans are highly negatively charged macromolecules due to their glycosaminoglycan side chains. The interaction of cationic probes with proteoglycans is of an electrostatic nature. Methods are discussed to increase the specificity of probes for proteoglycans. The use of phthalocyanin-like dyes such as Cuprolinic blue, according to the critical electrolyte concentration method, results in a selective staining of proteoglycans. Enzymatic or chemical digestions, however, should be done to validate the proteoglycan nature of the dye-positive granules/filaments, and to establish the class of proteoglycan. The value of cationic probes in basement membrane research on development and pathology is discussed. The potential for deducting molecular information from the ultrastructural appearance of stained proteoglycans is indicated.

Discrimination of the nucleolus by a critical electrolyte concentration method.

A new technique for the visualization of the nucleolus based on a variant of the previously reported critical electrolyte concentration method for nucleoprotein complexes is proposed. The method consists of treating toluidine blue-stained preparations with Mg2+ ions in a concentration such that ribonucleoprotein complexes are metachromatically highlighted, while DNA-protein complexes attain their critical electrolyte concentration point, thus losing metachromasy. The technique, which is applicable to plant and animal cells, is proposed for studies involving identification, morphometry and eventually changes in substructure of the nucleolus at the light microscopy level.

Effects of cis‐4‐hydroxy‐L‐proline on the androgen‐lnduced growth of the prostate of the prepubertally castrated guinea pig

The present study examined the effects of cis-4-hydroxy-L-proline (CHP), a proline analog, on the androgen-induced growth of the lateral prostate of prepubertally castrated guinea pigs. Prepubertal male guinea pigs were castrated at the age of 3 weeks and allowed to recover completely before subjection to an experimental regime to alter the stromal collagen synthesis by CHP. The animals were kept on a special proline-deficient diet (PDD) for a week before the subcutaneous injection of CHP (200 mg/kg/day) for 3 days, followed by a combined injection of CHP and dihydrotestosterone (DHT) (10 mg/kg/day) for 10 days. Control animals were injected with saline and DHT only. At the end of the experiment, the lateral prostate was removed and examined by 1) conventional transmission electron microscopy (TEM), 2) staining of proteoglycans (PGs) by Cuprolinic Blue (CB) using the critical electrolyte concentration (CEC) method, 3) carbohydrate and lectin histochemistry, and 4) electron microscopy (EM) lectin-gold labelling. The results showed that the wet weight of prostate from CHP-treated animals was significantly lower than the control and recovery groups. The epithelium was low columnar with an obvious increase in intercellular spaces and number of basal cells. The glandular cells showed little secretory activity with a decrease in number of granular endoplasmic reticulum (GER) profiles, secretory granules, and a small Golgi apparatus. The stroma was composed of stromal cells separated by large intercellular spaces with very sparse collagen fibrils, and a decrease in stromal PGs especially those PGs normally associated with collagen fibrils. CHP treatment also caused perturbation and disorganization in the epithelial basement membrane. The results suggested that stromal collagen is essential in mediating the response of glandular cells to DHT stimulation. Defective stromal collagens hamper the responsiveness of prostatic gland to androgen.

Concomitant changes in endothelial cell junctions and extracellular matrix components in the chick embryo aorta

According to previous studies, a process of endothelial activation seems to be occurring in the chick embryo between days 7 and 18. Also, endothelial cells respond to collagen as a substratum between 12 and 18 days, and this response diminishes until it almost disappears after birth. In the present study, aortas from chick embryos (days 7 to 21), and from chicks (14 days posthatching) were used. The results obtained by the freeze-fracturing technique, showed that between days 12 and 14 the intramembranous particles were aggregated into linear or clustered arrays in the fracture P-face of endothelial cells. This could signify that some kind of gap junction-like coupling may occur between adjacent endothelial cells. Our results also indicate that in advanced stages (21-day-old chick embryos and 14-day-old chicks) the growth of small aggregates into larger aggregates or plaques could occur. In addition to gap junctions, the presence of macular and linear tight junctions, reported as focal tight junctions (day 14 of development) macular and linear tight junctions with free-ending strands oriented parallel to one another (21 days) and smooth contoured ridges (14 days post-hatching) were observed. This sequence of changes may represent a development from linear to macular, to a more occluding arrangement, and may also reflect an endothelial cell polarization. Histochemical study of proteoglycans was done by using cuprolinic blue according to the critical electrolyte concentration method. Cuprolinic blue-positive granular, elongated and microfibrillar materials were found in the subendothelial region, forming a meshwork that occupies the extracellular space. Qualitative and quantitative changes were observed both in proteoglycans and in other extracellular matrix components throughout development, suggesting an increase in extracellular matrix complexity. These results lead us to suggest that the assembly of a more complex extracellular matrix, concomitantly with the formation of intercellular junctions during development, might influence the polarization of endothelium in the aorta of the chick embryo.

The use of cationic cacodylate iron colloid for the histochemical staining of acid mucopolysaccharides in human submandibular gland.

Both ferric hydroxide-cacodylate colloid (Fe-Cac) and a fine-grain one (Fe-Cac-f) are stable and carry positive charges over a wide range of pH. Using the colloid solution, some reliable information about the histochemical staining of acid mucopolysaccharides in human submandibular gland tissue could be obtained. Fe-Cac staining at pH 2 followed by Prussian blue reaction showed a diffuse blue staining of mucus acini, the intensity of which was equal to that of the staining at pH 4, clearly showing the sulfated mucopolysaccharides in the mucin. Most of serous acini were stained as blue granules in their cytoplasm. Fe-Cac-f staining gave a more distinct deep color by the Prussian blue reaction to the components, because of a superior diffusibility of the colloid into tissues. By Fe-Cac-f staining at pH 2 diffusely stained substances were found in the intercalated and striated ducts, showing the accumulation or passage of the secretions. The method will be useful for histochemical researchers in the oral field, in place of Alcian blue and its critical electrolyte concentration method.

Cytochemical characterization of cuprolinic blue-stained proteoglycans in the epithelial-stromal interface of the guinea pig lateral prostate.

Three types (T1, T2, T3) of proteoglycan (PG) filaments, as demonstrated by cuprolinic blue (CB) under critical electrolyte concentration method in the epithelial-stromal interface of the guinea pig lateral prostate, were characterized cytochemically by using a number of glycosaminoglycan(GAG)-degrading enzymes and nitrous acid. The results showed that T1 filaments located in basement membranes of the epithelium, endothelium, and smooth muscle cells, were removed by nitrous acid, heparitinase, and pronase but resistant to chondroitinase (Ch)-ABC and Ch-AC, heparinase, neuraminidase, and Streptomyces (S) hyaluronidase. The T1 filaments, therefore, contain heparan sulfate. The T2 filaments closely linked to collagen fibrils were removed by Ch-ABC, Ch-ABC plus S-hyaluronidase, and pronase but were resistant to nitrous acid, heparitinase, heparinase, neuraminidase, and S-hyaluronidase. These show that T2 filaments are rich in dermatan sulfate. The T3 filaments in the interstitial spaces and on the surface of fibroblasts were removed by Ch-ABC, Ch-AC, and pronase but were resistant to heparitinase, heparinase, hyaluronidase, neuraminidase, and nitrous acid. They are, therefore, rich in chondroitin sulfate.

Ultrastructural localization of proteoglycans by cationic dyes in the epithelial—stromal interface of the guinea pig lateral prostate

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.

Localization of proteoglycan within the extracellular matrix sheath of cone photoreceptors

The interphotoreceptor matrix of the human retina was examined histochemically by staining with a cationic copper phthalocyanin dye, Cuprolinic Blue, in a critical electrolyte concentration method which allowed staining of sulfated polyanions. In the presence of Cuprolinic Blue, a dense punctate matrix component was visualized covering the cone from the outer limiting membrane of the retina to the tip of the cone outer segment and extending through the matrix to the apical surface of the pigment epithelium. The size and appearance of the stained material are consistent with its being proteoglycan and its distribution is consistent with its being a component of the cone extracellular matrix sheath. A fine reticular pattern was observed in the presence and absence of Cuprolinic Blue.

Histological localization of myxoid tissue in normal human palatal mucosa and its glycosaminoglycans

Eighteen specimens of palatal mucosa were taken from 17 human subjects. Paraffin-wax sections were stained by routine methods and with various techniques to demonstrate glycosaminoglycans (GAG). In some sections, GAG were removed by selective degradative procedures before staining. Beneath all rugae, there were myxoid areas varying in size and marginal definition. Collagen fibres were few; elastic and reticulin fibres were numerous in a minority of sections. Alcianophilia at pH 2.5, preventable by streptomyces hyaluronidase digestion, suggested the presence of hyaluronic acid beneath the rugae. Alcian-blue staining at pH 1.0 and with the critical electrolyte concentration method using 0.5 M MgCl 2 did not distinguish the myxoid tissue from the surrounding connective tissue and could be prevented by digestion with testicular hyaluronidase or chondroitinase ABC. Chondroitin sulphate and, or dermatan sulphate thus may be present but were not localized to the myxoid tissue This unusual zone of loose connective tissue may act as a physical buffer resisting the local effects of high loads by allowing reversible extrusion of the water.

MULTIFOCAL ISCHAEMIC ENCEPHALOMYELOPATHY ASSOCIATED WITH FIBROCARTILAGINOUS EMBOLI IN THE LAMB

Multifocal, arterial, fibrocartilaginous embolism of the cervical spinal cord, medulla oblongata and cerebellum is described in a neonatal lamb. The emboli contained mucosubstances which, by the Alcian blue critical electrolyte concentration method, were identified as mainly keratan sulphate. This composition indicates the probable origin of emboli from the nucleus pulposus of intervertebral discs. Possible modes of entry of such material into the spinal cord vasculature are reviewed and a mechanism proposed to explain the localization of emboli in spinal cord, brain stem and cerebellar arteries in this case.

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites.

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.

Histochemical detection of anionic components in the cephalopod brain

Brains obtained from three species of mediterranean cephalopods (Loligo, Sepia, Octopus) were fixed in Bouin's solution. Paraffin sections were cut sequentially at the frontal plane and used for Alcian blue staining (critical electrolyte concentration method), colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method, and the lead tetra-acetate-Schiff method. The stained sections were evaluated at 2 regions of different histological composition: the palliovisceral ganglion and the optic lobe. A high concentration of anionic components was found in synaptic regions of the neuropil whereas neuronal cell bodies showed a relatively weak staining of these constituents. There was a significant reaction of the perineuronal glia nets in the cellular rind of the palliovisceral ganglion. From the comparison of staining patterns obtained with the 4 methods in this study and literature data it can be concluded that the detected anionic sites are mainly carboxyl groups of acidic proteins and/or glycoproteins. Sulphate groups may be present in lower concentrations. Their distribution reveals that the role of anionic components other than sialic acids in the invertebrate brain might be discussed in the context of synaptic transmission similar to that in vertebrates. The possible involvement of the glia cell population has to be taken into consideration.

Proteoglycan association with collagen d band in hyaline articular cartilage.

Proteoglycans of canine articular cartilage were labelled for transmission electron microscopy using the cationic copper phthalocyanin dye, cupromeronic blue, in a critical electrolyte concentration method. Much of the proteoglycan appeared to be structurally unrelated to collagen but a small proportion was positioned close to fibrils. On demonstrating the characteristic collagen banding pattern with uranyl acetate and phosphotungstic acid, it was evident that proteoglycan interacted with collagen at the d band.

Wheat germ agglutinin indicates the commencement of neuronal differentiation in the embryonic brain. A comparative carbohydrate histochemical study

Paraffin sections prepared from prenatal rat brain (embryonic days 14, 16, 18 and 20) were incubated with buffered wheat germ agglutinin labelled with fluorescein isothiocyanate (FITC-WGA). In the embryonic brain, the proliferating cells of the ventricular zone showed a low binding of WGA. Differentiating neuroblasts and axonal bundles exhibited a significantly higher degree of labelling. This pattern was most evident in the cerebral cortex where the marginal zone and the sub-plate region were found to be most intensely strained. The structural basis of WGA-binding was specified by comparison with data obtained by the colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method and Alcian Blue staining (critical electrolyte concentration method). The results show that at regions of intensive differentiation in the embryonic brain there is an increase in the concentrations of sialic acid and glycosaminoglycans.