The proteoglycan skeleton of the Kurloff body as evidenced by Cuprolinic Blue staining

Summary 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.     

Staining of proteoglycans in mouse lung alveoli. II. Characterization of the Cuprolinic Blue-positive, anionic sites

Summary The nature of Cuprolinic Blue-positive anionic filaments in mouse lung alveoli has been characterized. The contrast of filaments in the alveolar basement membrane of type I epithelial cells was lost on treatment with nitrous acid and pronase (without prefixation). In contrast, neither neuraminidase, chondroitinase ABC or AC, norStreptomyces hyaluronidase had any effect. Treatment with pronase (after prefixation) and 2.0m MgCl₂ (after prefixation) also had no effect, indicating that the filaments are heparan sulphate proteoglycans. The filaments in the alveolar basement membrane of type II epithelial cells and in the capillary basement membrane of the endothelial cells were also nitrous acid sensitive, but chondroitinase ABC-insensitive. A model in which the whole alveolus contains a single layer of heparan sulphate-containing proteoglycan monomers is proposed. Furthermore, the collagen fibril associated filaments remained unaffected after treatment with nitrous acid, neuraminidase orStreptomyces hyaluronidase, or after digestion with pronase (after prefixation) and treatment with 2.0m MgCl₂ (after prefixation). These filaments, however, could no longer be detected when digestion with chondroitinase ABC or pronase (without prefixation) was applied; chondroitinase AC treatment clearly affected the filaments, although they still were visible. These results indicate that the filaments are dermatan sulphate-containing proteoglycans. Some functional aspects of the proteoglycans are discussed.     

Proteoglycans in arterial smooth muscle cell cultures: an ultrastructural histochemical analysis

The extracellular matrix in cultures of arterial smooth muscle cells has been examined by ultrastructural histochemistry using each of the following cationic dyes: ruthenium red, Alcian blue, acridine orange, and safranin O. All dyes exhibited an affinity for a structural component that was either preserved as a granule with ruthenium red or Alcian blue, or as an extended filament or bottlebrush structure with acridine orange or safranin O. Both granules and filaments were removed when the cultures were pretreated with chondroitinase ABC, an enzyme that degrades the glycosaminoglycan moiety of some proteoglycans. These structural components of the extracellular matrix were not observed when cultures were prepared in the absence of the cationic dyes. Labeling experiments (35S-sulfate) revealed that approximately 40% of the total labeled proteoglycans were lost during routine processing for electron microscopy (i.e., fixation through dehydration). Inclusion of any one of the cationic dyes during fixation reduced the losses to less than 1%. The extended filamentous structure preserved by safranin O and acridine orange resembled the structure of purified proteoglycans prepared from the same cultures and spread on cytochrome c monolayer films. These observations suggest that proteoglycans exist as extended bottlebrush structures within the extracellular matrix, and support the interpretation that the granular deposits observed in the ruthenium red and Alcian blue preparations most likely represent individual proteoglycan monomers that have undergone molecular collapse during processing. In addition, the dyes also exhibited an affinity for chords of fine fibrils that contained small granules and/or filaments. Both the fibrillar material and the associated granular and filamentous structures enmeshed in the fibrils resisted digestion with chondroitinase ABC.     

Experimental mucopolysaccharidosis: Preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue

Summary The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl₂ simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.     

Ultrastructural localization of keratinocyte surface associated heparan sulphate proteoglycans in human epidermis

Summary Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens containd round granules ( 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl₂ indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC, Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the ³⁵SO₄ labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/ dermatan sulphates comprised about one fifth of the total activity incorporated. The distribution of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.     

Differential staining of glycosaminoglycans in the predentine and dentine of rat incisor using Cuprolinic Blue at various magnesium chloride concentrations

Summary Rat incisors were fixed with a solution of 0.05% Cuprolinic Blue and 2.5% glutaraldehyde in the presence of various concentrations of MgCl₂ according to the critical electrolyte concentration (CEC) principle. This method allows glycosaminoglycans (GAG) to be properly preserved and visualized. Small granules were stained by the cationic dye in the predentine in the absence of MgCl₂. These granules grew in size and became more electron-dense when the concentration of the electrolyte was increased. Larger ribbon-like structures and granules were seen when 0.3M MgCl₂ was used. In the dentine, tiny dots in close association with the surface of the collagen fibres, or their periodic striations, were positively stained. A thick electron-dense band located on the dentine side at the predentine-dentine junction was seen both with and without 0.05 M MgCl₂. With higher concentrations of the electrolyte (0.1–0.3 M), this band was reduced to a very thin line located at the border of the dentine, along with mineralizing collagen fibres. This demonstrated the presence of GAG at the dentine surface and therefore indicated that GAG may play a role as nucleator agent.     

Sulfated glycosaminoglycans in guinea pig basophils studied by means of cationic colloidal gold

 Bone marrow embedding in the hydrophilic resin, Lowicryl K4M, followed by cationic colloidal gold (CCG, pH 1.0) staining was used to study the sulfated glycosaminoglycans (GAGs) and their sites of sulfation ultrastructurally in various maturational stages of both basophil granulocytes and basophil granules in the guinea pig. CCG at pH 1.0 is specific for sulfated GAG staining. Basophil granulocytes and granules reacted positively to CCG with a variety of staining according to the stage of maturation. The formation of basophil granules takes place throughout the myelocyte stage. Early basophil myelocytes contain a large Golgi apparatus with active granulogenesis, while late myelocytes contain a small and less active Golgi apparatus as judged by CCG staining. All the immature granules and some of the granules with characteristic ultrastructure stained positively. However, some of the mature granules had lost their affinity for CCG upon maturation. Interestingly, strongly positive CCG staining was also observed in the trans to transmost Golgi apparatus. This indicates that sulfation of GAGs occurs in the trans to transmost Golgi apparatus in all maturational stages of basophil granulocytes. Treatment with chondroitinase ABC or heparinase I abolished the majority of CCG staining. Accepted: 17 July 1997     

Proteoglycans in primate arteries. I. Ultrastructural localization and distribution in the intima.

Proteoglycans were identified and localized histochemically and ultrastructurally in normal and hyperplastic arterial intimas in nonhuman primates (Macaca nemestrina). These regions were consistently more alcianophilic than the adjacent medial layers and this alcianophilia was absent after treatment with glycosaminoglycan-degradative enzymes. Ultrastructurally, the intimal intercellular matrix consisted of numerous, irregularly shaped, 200-500-A diameter granules possessing 30--60-A diameter filamentous projections, and these granules were dispersed between collagen and elastic fibers. The granules exhibited a marked affinity for ruthenium red and were interconnected via their filamentous projections. The ruthenium red-positive granules were intimately associated with the plasma membrane of intimal smooth muscle cells and attached to collagen fibrils and elastic fibers. The matrix granules were completely removed after testicular hyaluronidase or chondroitinase ABC digestion but only partially removed after leech hyaluronidase treatment. These results suggest that the matrix granules contain some hyaluronic acid and one or more isomers of chondroitin sulfate. In addition to the large ruthenium red-positive matrix granules, a smaller class of ruthenium red-positive granule (100--200-A diameter) was present within the basement membranes beneath the endothelium and surrounding the smooth muscle cells. Ruthenium red also exhibited an affinity for the surface coat of the smooth muscle cells. The potential importance of proteoglycans in arterial intimal hyperplasia is discussed.     

Specific binding of peanut agglutinin and soybean agglutinin to chondroitinase ABC-digested cartilage proteoglycans: Histochemical, ultrastructural cytochemical, and biochemical characterization

Summary The binding of peanut agglutinin (PNA) and soybean agglutinin (SBA) to cartilage proteoglycans was investigated by histochemical, ultrastructural cytochemical, and biochemical methods. Following aldehyde fixation, specimens of rat epiphyseal cartilage were examined by horseradish peroxidase-labelled lectin cytochemistry with and without prior digestion in chondroitinase ABC. At the light microscope level neither PNA nor SBA exhibited any affinity for cartilage matrix, but became strongly bound following chondroitinase treatment. Similarly, at the ultrastructural level, extracellular matrix granules, presumed to be proteoglycan monomer(s), lacked PNA affinity in undigested specimens, and stained very weakly with SBA. Both PNA and SBA weakly to moderately stained thetrans cisternae of the Golgi-flattened cisternae in chondrocytes. The chondrocyte plasmalemma lacked PNA staining, but reacted weakly with SBA. Following chondroitinase digestion, PNA and SBA stained matrix granules, and the cell surface of chondrocytes intensely, whereas the Golgitrans cisternae, the Golgi-derived vacuoles, and multivesicular bodies demonstrated weak to moderate reactivity. Proteoglycan aggregates purified from rat chondrosarcoma and bovine nasal cartilage bound PNA and SBA avidly after digestion with chondroitinase. Undigested proteoglycans lacked affinity for PNA and reacted very weakly with SBA. These results indicate that both PNA and SBA specifically react with chondroitinase-modified oligosaccharide(s) bound to core proteins of cartilage proteoglycans. This provided a specific histochemical and ultrastructural cytochemical procedure for localizing chondroitin sulphate-containing proteoglycans.     

Polyethyleneimine as a contrast agent for ultrastructural localization and characterization of proteoglycans in the matrix of cartilage and bone.

We examined the presence of proteoglycans in the extracellular matrix of cartilage and bone in fetal mouse radii at the ultrastructural level, using the cationic dye polyethyleneimine (PEI). After staining with this dye, the proteoglycans appeared as granules in the uncalcified bone matrix and as extended winding structures in the cartilage matrix. PEI-positive material was removed after treatment of the tissue with chondroitinase ABC. Inhibition of the proteoglycan synthesis by beta-D-xyloside resulted in smaller PEI-positive windings in the cartilage matrix. These observations suggest that the winding, PEI-positive structures represent proteoglycan aggregates. No loss of PEI-positive material in the calcified cartilage matrix was seen, suggesting that proteoglycans do not need to be removed to make the matrix calcifiable.     

Further characterization of a large proteoglycan in human lung alveoli

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, heavily staining, generally large, filaments have been demonstrated in human lung alveoli. In some lung specimens they are abundant, while in others they are very scanty. The filaments are seen: around bundles of collagen fibrils, at places which seem electron microscopically almost empty, associated with basement membranes around elastin, and sometimes associated with individual collagen fibrils. After poststaining tiny threads--connecting the filaments--could sometimes be observed. The filaments are resistant to treatment with nitrous acid, heparitinase or pronase after prefixation. After digestion with chondroitinase ABC, chondroitinase AC or pronase without prefixation, the filaments are no longer detectable. The tiny threads are chondroitinase ABC resistant. It is concluded that the Cuprolinic Blue-positive filaments represent proteoglycans which contain chondroitin sulfate and/or glucuronic acid-rich dermatan sulfate. The possible role of these proteoglycans in tissue repair is discussed.     

Quantitative X-ray microanalysis of adrenal medullary cells of young adult and aged rats after glutaraldehyde fixation and potassium dichromate treatment

X-ray microanalysis has been used to detect chromium in the histochemical reaction product resulting from the reaction of noradrenaline with glutaraldehyde during fixation of the rat adrenal medulla and subsequent treatment with potassium dichromate. In unstained ultrathin sections, noradrenaline cells can be identified by their content of highly electron-dense storage granules, which enables individual granules to be analysed quantitatively to assess the amount of bound chromium within them. In young adult (4-month-old) rats the mean chromium content of noradrenaline-containing adrenal medullary granules was 443.6±50.7 mM/kg dry weight. In aged (24-month-old) animals the mean chromium content was 267.0±64.0 mM/kg dry weight which was significantly (P<0.01) lower then the value for the young adult rats. Some noradrenaline cells contained granule populations, which were markedly less electron dense than those in the young adults and this is reflected in the ranges of chromium values recorded between individual cells in the 24-month-old animals. There were also noradrenaline cells in the medulla of the aged animals, which contained highly electron-dense granules but these did not contain as much bound chromium as the highest values recorded in the young adult animals. The results are discussed in the context of the growth of the rat adrenal medulla throughout the lifespan and with respect to the effects of age on the integrity of storage granules.     

Chondroitin sulfate proteoglycan is a constituent of the basement membrane in the rat embryo parietal yolk sac

Summary In addition to containing Type IV collagen, laminin and entactin, basement membranes contain small amounts of proteoglycans substituted primarily with heparan sulfate chains. We have previously shown, however, that parietal yolk sacs in organ culture synthesize predominantly chondroitin sulfate proteoglycan. In the present study, we have used histochemical and immunohistochemical techniques coupled with chondroitinase ABC digestion to provide evidence for the presence of chondroitin sulfate proteoglycan in the basement membrane (Reichert's membrane) of the 14.5-day rat embryo parietal yolk sac. The results revealed numerous cuprolinic blue-positive filaments and granules, 20–30 nm in greater length or diameter, dispersed throughout the thickness of the basement membrane. Both structures were removed by preincubating freshly isolated parietal yolk sacs with chondroitinase ABC. A similar labeling pattern was also obtained with immunoelectron microscopy using gold-labeled monoclonal anti-bodies directed against the three major isomers of protein-bound chondroitin sulfate. In contrast, coarser cuprolinic blue granules, 40–100 nm in diameter, were neither sensitive to chondroitinase ABC digestion nor labeled by the monoclonal antibodies. These results thus indicate that Reichert's membrane contains chondroitin sulfate proteoglycan in addition to heparan sulfate proteoglycan.     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study

Summary The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 m in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled. From these findings, it is suggested that the granules of rat Clara cells consist of two types of granules of distinct origin; one appears to derive from condensing vacuoles of Golgi origin, whereas the other may be formed by membranefusions with small cytoplasmic vesicles of unknown source.     

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky’s fixative and OsO₄ and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.     

Immunoelectron microscopic study of proteoglycans in rat epiphyseal growth plate cartilage after fixation with ruthenium hexamine trichloride (RHT).

The localization of proteoglycans in rat epiphyseal growth plate cartilage was investigated immunoelectron microscopically by the post-embedding method, using mouse monoclonal antibody (2-B-6) which specifically recognizes 4-sulphated chondroitin or dermatan sulphate after digestion of proteoglycans with chondroitinase ABC. Fixation with ruthenium hexamine trichloride (RHT) and embedding in LR White served to preserve chondrocytes in the expanded state and matrix proteoglycans were observed as a reticular network of filaments. Immunoelectron microscopy revealed gold labelling of the secondary antibodies for the demonstration of proteoglycans on these filamentous structures and in elements of the Golgi apparatus. Filaments associated with matrix vesicles were also labelled. After fixation in the presence of RHT, it was clearly demonstrated that cartilage matrix proteoglycans are retained approximately in their original spatial distribution and their antigenicity is well preserved.     

Immunoelectron microscopic study of proteoglycans in rat epiphyseal growth plate cartilage after fixation with ruthenium hexamine trichloride (RHT)

The localization of proteoglycans in rat epiphyseal growth plate cartilage was investigated immunoelectron microscopically by the post-embedding method, using mouse monoclonal antibody (2-B-6) which specifically recognizes 4-sulphated chondroitin or dermatan sulphate after digestion of proteoglycans with chondroitinase ABC. Fixation with ruthenium hexamine trichloride (RHT) and embedding in LR White served to preserve chondrocytes in the expanded state and matrix proteoglycans were observed as a reticular network of filaments. Immunoelectron microscopy revealed gold labelling of the secondary antibodies for the demonstration of proteoglycans on these filamentous structures and in elements of the Golgi apparatus. Filaments associated with matrix vesicles were also labelled. After fixation in the presence of RHT, it was clearly demonstrated that cartilage matrix proteoglycans are retained approximately in their original spatial distribution and their antigenicity is well preserved.     

Removal of proteoglycans increases efficiency of retroviral gene transfer

We have previously shown that medium conditioned by virus producer cells inhibits retrovirus transduction, and that a portion of the inhibitory activity is sensitive to chondroitinase ABC. In this study, we have quantitatively evaluated the fraction of the inhibitory activity that is due to chondroitinase ABC-sensitive material and partially characterized the inhibitors. The kinetics of chondroitinase ABC digestion of glycosaminoglycans and virus inhibitory activity in cell culture medium were measured, and the results used to estimate the amount of the chondroitinase ABC-sensitive virus inhibitory activity that was initially in the medium. We found that up to 76% of the inhibitory activity of medium conditioned by packaging cells derived from NIH 3T3 cells is sensitive to chondroitinase ABC. The remainder of the inhibitory activity is not sensitive to other glycosaminoglycan lyases (heparitinase I or heparinase I), which suggests that substances other than glycosaminoglycans or proteoglycans are present in virus stocks and inhibit transduction. To further characterize the inhibitors, proteoglycans from conditioned medium were purified by batch anion exchange and size exclusion chromatography. Two major size groups (100 kDa and 950 kDa) of proteoglycans were isolated. Transduction was inhibited 50% by 0.6 microg/mL of the high-molecular-weight proteoglycan or by 1.7 microg/mL of the low-molecular-weight proteoglycan. Significantly, the proteoglycans, because of their large size and poor sieving properties, coconcentrated with virus particles concentrated by ultrafiltration and prevented any significant increases in transduction efficiency. Transduction efficiencies of virus stocks were increased more than tenfold by ultrafiltration, but only when the concentrated virus was treated with chondroitinase ABC.     

Morphological Characterization of Three Phenotypes of the Isopod Armadillidium vulgare

The morphological characteristics and ommochrome quantity in the integument of red, white, and wild type (black-grey) Armadillidium vulgare were studied. The red phenotype was found to possess two kinds of immature ommochrome pigment granules within its pigment cells, in addition to mature pigment granules. The immature granules seemed to contain uniformly distributed fibrilles, or to have an electron-dense central region surrounded by an electron-lucent outer edge. Since these immature pigment granules were typically observed to be distributed along with the mature ones, and were also more easily extractable than the wild type's, it is hypothesized that ommochrome granule maturation in the red phenotype may occur slowly due to a defect in the pigment granule internal process which combines pigments with matrix proteins. Regarding the white phenotype, although its pigment cells were undeveloped, several large-sized vesicles containing a small amount of electron-dense material appeared in the pigment cell cytoplasm. The wild and red type males of A. vulgare were found to have an ommochrome content twice as large as that of the corresponding females, with no ommochrome pigment being detected in the white phenotype. The genetic relationship between the white and red phenotypes was discussed using as a basis the observed pigment granule structure.     

Ultrastructural localization of keratinocyte surface associated heparan sulphate proteoglycans in human epidermis

Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens contained round granules (luminal diameter 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl2 indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC. Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the 35SO4 labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/dermatan sulphates comprised about one fifth of the total activity incorporated. The distributions of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.