Electron microscopy of cartilage proteoglycans

Synopsis The proteoglycans of cartilage are complex molecules in which chondroitin sulphate and keratan sulphate chains are covalently linked to a protein core, forming a polydisperse population of proteoglycan monomers. By interaction with hyaluronic acid and link proteins, the monomers form large macromolecular complexes.In vivo the proteoglycans mainly occur in such aggregates. In the electron microscope, the cartilaginous matrix can be seen to be made up of thin collagen fibrils and polygonal granules about 10–50 nm in diameter. Addition of the polyvalent cationic dye Ruthenium Red to glutaraldehyde and osmium tetroxide fixatives yields a dense selective staining of the matrix granules. Following a short digestion of cartilage slices with either of the chondroitin sulphate-degrading enzymes hyaluronidase and chondroitinase or with the proteolytic enzyme papain, the matrix granules were few in number or completely absent and the proteoglycan content, measured as hexosamine, decreased by up to 90%. Similarly, extraction of the cartilage with 4 M guanidine-HCl removed all matrix granules and most of the proteoglycans. From these findings, it can be concluded that the matrix granules represent proteoglycans, most probably in aggregate form, and that Ruthenium Red staining may be used to study the distribution of these macromolecules in thin sections. As a complement to chemical studies on proteoglycan structure, it is also possible to observe and measure individual molecules in the electron microscope after spreading them into a monomolecular layer with cytochromec. This technique has been applied in investigations on proteogly cans isolated from bovine nasal cartilage and other hyaline cartilages. The molecules in the monomer fractions appeared as an extended central core filament to which about 25–30 side-chain filaments were attached at various intervals. The core filament, averaging about 300 nm in length, was interpreted as representing the polysaccharide-binding part of the protein core and the side-chain filaments, averaging about 45 nm in length, as representing the clusters of chondroitin sulphate chains. Statistical treatment of the collected data indicated that no distinct subpopulations existed within the monomer fractions. The electron microscopic results correlated well with chemical data for the corresponding fractions and together with recent observations on various aggregate fractions strongly support present concepts of proteoglycan structure.Paper presented at a symposium The Changing directions of carbohydrate histochemistry at the Fifth International Congress of Cytochemistry and Histochemistry in Bucharest, Romania on September 1976.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

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

Summary 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 method, 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 m-thick paraffin and 1 m-thick glycolmethacrylate sections of bovine articular cartilage. The staining intensity of the sections was measured from spots 25 m 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 m-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.     

Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.

The glycosaminoglycans of human cultured normal glial and malignant glioma cells were studied. [35S]Sulphate or [3H]glucosamine added to the culture medium was incorporated into glycosaminoglycans; labelled glycosaminoglycans were isolated by DEAE-cellulose chromatography or gel chromatography. A simple procedure was developed for measurement of individual sulphated glycosaminoglycans in cell-culture fluids. In normal cultures the glycosaminoglycans of the pericellular pool (trypsin-susceptible material), the membrane fraction (trypsin-susceptible material of EDTA-detached cells) and the substrate-attached material consisted mainly of heparan sulphate. The intra- and extra-cellular pools showed a predominance of dermatan sulphate. The net production of hyaluronic acid was low. The accumulation of 35S-labelled glycosaminoglycans in the extracellular pool was essentially linear with time up to 72h. The malignant glioma cells differed in most aspects tested. The total production of glycosaminoglycans was much greater owing to a high production of hyaluronic acid and hyaluronic acid was the major cell-surface-associated glycosaminoglycan in these cultures. Among the sulphated glycosaminoglycans chondroitin sulphate, rather than heparan sulphate, was the predominant species of the pericellular pool. This was also true for the membrane fraction and substrate-attached material. Furthermore, the accumulation of extracellular 35S-labelled glycosaminoglycans was initially delayed for several hours and did not become linear with time until after 24 h of incubation. The glioma cells produced little dermatan sulphate and the dermatan sulphate chains differed from those of normal cultures with respect to the distribution of iduronic acid residues. The observed differences between normal glial and malignant glioma cells were not dependent on cell density; rather they were due to the malignant transformation itself.     

Distribution of proteoglycans and hyaluronic acid in transverse sections of bovine thoracic aorta

Summary The distribution of hyaluronic acid and proteoglycans in bovine thoracic aorta was studied by Alcian Blue staining of frozen tissue sections under controlled electrolyte conditions with and without prior enzymic digestion. Some sections were digested with chondroitinase ABC, testicular hyaluronidase or bacterial collagenase and subsequent staining permitted conclusions to be drawn about the distribution of specific glycosaminoglycans within the tissue. The total glycosaminoglycan content was maximal in the intima and decreased across the arterial wall to the outermost adventitial layer. The content of proteoglycan containing chondroitin sulphate and/or dermatan sulphate chains paralleled this distribution. However, other glycosaminoglycans also contributed significantly to staining, although there was no evidence for any appreciable concentration of heparin or highly sulphated heparan sulphate.Several experiments indicated that proteoglycan containing chondroitin sulphate and/or dermatan sulphate was associated with elastic laminae which were often seen stained along their periphery. Hyaluronic acid was present at significant concentrations in all locations of the aorta and there was evidence for a similar distribution of heparan sulphate which was possibly also present at a high concentration in the endothelium. Staining of sections after treatment with 4m guanidinium chloride confirmed that this extractant removed most of the proteoglycan from the tissue section.     

Localization of proteoglycan monomer and link protein in the matrix of bovine articular cartilage: An immunohistochemical study

Using monospecific antisera and immunofluorescence microscopy, proteoglycan monomer (PG), and link proteins were demonstrated throughout the extracellular matrix of bovine articular cartilage. A narrow band of strong pericellular staining was usually observed for both molecules, indicating a pericellular concentration of proteoglycan monomer: this conclusion was supported by dye-binding studies. Whereas PG was evenly distributed throughout the remaining matrix, more link protein was detectable in interterritorial sites in middle and deep zones. Well-defined zones of weaker territorial staining for link protein stained strongest for chondroitin sulfate. Trypsin treatment of cartilage resulted in a loss of most of the PG staining, but some selective retention of link protein, particularly around chondrocytes in the superficial zone at and near the articular surface. This residual staining was largely removed if sections were fixed after chondroitinase treatment. After extraction of cartilage with 4M guanidine hydrochloride, only PG remained and this was concentrated in the superficial zone. These observations are shown to support the concept of aggregation of PG and link protein with hyaluronic acid (HA) in cartilage matrix, and the binding of PG and link protein to HA, which is attached to the chondrocyte surface. Culture of cartilage depleted of PG and link protein by trypsin demonstrated that individual chondrocytes can secrete both PG and link proteins and that the organization of cartilage matrix can be regenerated in part over a period of 4 days.     

Demonstration of chondroitin sulphate and glycoproteins in articular cartilage matrix using periodic acid-Schiff (PAS) method

Summary Staining of articular cartilage by the periodic acid-Schiff (PAS) method was measured using microspectrophotometry. Standard PAS technique with 2 h oxidation produced a distinct Schiff reaction in the cartilage sections. The staining increased with depth of the articular cartilage demonstrating distribution of the glycoproteins. The modified PAS method included a second, longer periodic acid treatment, which made the uronic acid of glycosaminoglycans PAS-positive. The modified PAS method proved to be highly specific for chondroitin sulphate, which was determined from the samples with gas chromatography. A statistically significant correlation between the Schiff reactivity and galactosamine content of the sections was observed. It is concluded that for articular cartilage standard and modified PAS methods are useful procedures for demonstrating local changes of glycoproteins and chondroitin sulphate, respectively.     

Demonstration of fibronectin in human articular cartilage by an indirect immunoperoxidase technique

Summary Fresh frozen tissue sections of human articular cartilage was treated without and with human testicular hyaluronidase (2×10⁶ units/l) for 60 min at 37° C and stained by the indirect immunoperoxidase technique with rabbit antihuman fibronectin. The rabbit antihuman fibronectin was purified by affinity chromatography on human fibronectin-Sepharose. Fibronectin was only found on the acellular surface of the articular cartilage in tissue sections not treated with hyaluronidase. In this surface layer, probably identical to lamina splendens, the arrangement of fibronectin was as a membrane. No collagen was seen in this area by van Gieson staining. No staining for fibronectin was found in the cartilage matrix or in the chondrocytes. Treatment of the cartilage tissue with hyaluronidase resulted in visualization of high amount of fibronectin in the cartilage matrix, with the highest intensity around the chondrocytes. The staining of the acellular surface layer of the articular cartilage was identical with the results obtained without hyaluronidase treatment. These results indicate that articular cartilage is rich in fibronectin probably in complex with hyaluronic acid, and that the chondrocytes produce fibronectin in situ. It also demonstrates the steric hindrance of hyaluronic acid aggregates in diffusion of the antibody and the value of hyaluronidase treatment of tissue before demonstration of fibronectin.     

Electron microscopic investigations on aging and osteoarthrotic human articular cartilage

Summary The submicroscopic morphology of freeze-etched aging human articular cartilage is reported. In contrast to chemically fixed and thin sectioned cartilage tissue, chondrocytes and matrix of freeze-etched specimens exhibit new morphological aspects.The surface of superficial chondrooytes shows invaginations and bulges, producing a cauliflower-like appearance of the cell body. Finger-like protrusions as found in thin sections are absent. The matrix adjacent to the pericellular halo, the latter consisting of a fine granular material, is characterized by the presence of globular, membrane-bounded vesicles of variable size. This vesicle containing zone is called corona. The corona vesicles as well as the cellular membranes are occupied by two types of particles (85 and 135 Å in diameter). Based on the presence of these particles, the nature and possible origin of the corona vesicles are discussed.     

Distribution of hyaluronan in articular cartilage as probed by a biotinylated binding region of aggrecan

The proportion of total tissue hyaluronan involved in interactions with aggrecan and link protein was estimated from extracts of canine knee articular cartilages using a biotinylated hyaluronan binding region-link protein complex (bHABC) of proteoglycan aggregate as a probe in an ELISA-like assay. Microscopic sections were stained with bHABC to reveal free hyaluronan in various sites and zones of the cartilages. Articular cartilage, cut into 20 m-thick sections, was extracted with 4 M guanidinium chloride (GuCl). Aliquots of the extract (after removing GuCl) were assayed for hyaluronan, before and after papain digestion. The GuCl extraction residues were analyzed after solubilization by papain. It was found that 47–51% of total hyaluronan remained in the GuCl extraction residue, in contrast to the 8–15% of total proteoglycans. Analysis of the extract revealed that 24–50% of its hyaluronan was directly detecable with the probe, while 50–76% became available only after protease digestion. The extracellular matrix in cartilage sections was stained with the bHABC probe only in the superficial zone and the periphery of the articular surfaces, both sites known to have a relatively low proteoglycan concentration. Trypsin pretreatment of the sections enhanced the staining of the intermediate and deep zones, presumably by removing the steric obstruction caused by the chondroitin sulfate binding region of aggrecans. Enhanced matrix staining in these zones was also obtained by a limited digestion with chondroitinase ABC. The results indicate that a part of cartilage hyaluronan is free from endogenous binding proteins, such as aggrecan and link protein, but that the chondroitin sulfate-rich region of aggrecan inhibits its probing in intact tissue sections. Therefore, hyaluronan staining was more intense in cartilage areas with lower aggrecan content. A large proportion of hyaluronan resists GuCl extraction, even from 20-m-thick tissue sections.     

Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions

Summary The application of the critical electrolyte concentration (CEC) concept to the differentiation of acidic glycosaminglycans (mucopolysaccharides) is described. Alcian Blue 8GX stains with increasing selectivity as increasing amounts of magnesium chloride are incorporated into the dye solution. Model experiments with pure polyanions, or artifically carboxylated, phosphorylated and sulphated liver sections, showed that binding of dye to carboxylate or phosphate groups ceased at low electrolyte concentrations (< 0.3M) whereas dye continued to be held by sulphate ester groups at concentrations five to ten times as high. The similarity to the well established cetylpyridinium system for polyanion fractionation is discussed.Sections of tissues chosen to contain predominantly or characteristically carboxylated mucins, and/or sulphate ester polyanions showed a staining pattern entirely similar to the model sections. Goblet cell mucin in rat ileum stained at < 0.4M MgCl₂, Cartilage at < 0.6M MgCl₂, mast cells at < 0.75M, and corneal stroma at < 1.0M. These results are in agreement with the known contents of sialo-mucin, chondroitin sulphate, heparin and keratansulphate, respectively. The conditions in which this principle can be used in a practical technique are described.The new and more precise terminology of Jeanloz (1960) is used in preference to the older nomenclature.     

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.     

Ultrastructural localization of acidic glycoconjugates with the low iron diamine method

The present study has applied the low iron diamine (LID) method at the ultrastructural level to demonstrate acid glycoconjugates. We have examined rat epiphyseal cartilage, human bone marrow, rat tracheal glands, and mouse sublingual glands stained with LID prior to embedment. The LID staining appeared to require postosmication for adequate visualization at the electron microscope level. Thiocarbohydrazide-silver proteinate (TCH-SP) staining of thin sections variably enhanced LID reactive sites. LID-TCH-SP stained carboxyl and sulfate groups of glycosaminoglycans in the extracellular cartilage matrix, secretory granules, and expanded Golgi saccules of chondrocytes. In human bone marrow, LID-TCH-SP variably stained the cytoplasmic granules, known to contain sulfated glycosaminoglycans, and the external surface of the plasma membrane of leukocytes. Moderately strong LID staining was observed in secretory granules in mucous tubules of rat tracheal glands, known to contain sulfated glycoproteins, and in acinar cells of mouse sublingual glands, known to contain a sialoglycoprotein. The lack of sulfated glycoconjugates in acinar cells of the mouse sublingual gland was confirmed by their failure to stain with the high iron diamine method. Thus these studies indicate that the LID and LID-TCH-SP methods are useful for the ultrastructural localization of carboxylated and sulfated glycoconjugates in extracellular and intracellular sites.     

Binding of wheat germ agglutinin in the matrix of rat tracheal cartilage

Summary The binding of wheat germ agglutinin (WGA) to the extracellular matrix of rat tracheal cartilage was studied at both the light and electron microscopic levels. The detection of binding sites was accomplished by a postembedment method using the direct fluorescence technique for light microscopy and the avidin—biotin bridging system for electron microscopy. Distinct fluorescence was observed in the pericellular region of chondrocytes, and this fluorescence was completely removed after treatment with 4 M guanidine hydrochloride. By electron microscopy, the reaction products as found in the pericellular region were not observed in the interterritorial collagenous matrix, confirming a similar distribution as found by fluorescence microscopy. These results show that WGA-binding sites are present in pericellular matrical substances, which are known to be rich in proteoglycan and glycosaminoglycan complexes and which exhibit similar staining with antibodies to proteoglycans or with cationic dyes. As WGA binds toN-acetyl-glucosamine andN-acetyl-neuraminic acid residues, the pericellular matrix of rat tracheal cartilage appears to consist of proteoglycan having a high concentration of these saccharides.     

Ultrastructural cytochemistry of oxytalan fibres in monkey periodontal ligaments with the high iron diamine method

Summary Monkey periodontal ligaments have been examined at the ultrastructural level to demonstrate the nature of reactive sites in oxytalan fibres. The high iron diamine (HID) and HID-thiocarbohydrazide-silver proteinate methods specific for sulphate groups, with and without prior oxidation with monopersulphate, were used. Oxytalan fibres were composed of bundles of microfibrils with a diameter of 11.5 ± 1.7 nm (mean ±s.d.,n = 50). In cross section the microfibrils were found to have a denser periphery, giving them a tubular appearance. The oxytalan microfibrils of non-oxidized specimens showed little reactivity with either HID method, except that the extracellular matrix material in close association with collagen fibrils stained weakly; in oxidized specimens, both HID methods strongly stained oxytalan microfibrils and weakly stained the extracellular matrix material. Such reactivity of oxytalan microfibrils was not altered by digestion with testicular hyaluronidase or chondroitinase ABC, performed prior to or after persulphate oxidation. Further, the sequential thiosulphation and HID method for the demonstration of disulphide and sulphhydryl groups stained oxytalan fibres moderately. These results indicate that the oxidative generation of sulphate groups in oxytalan fibres may occur from either disulphide or sulphhydryl groups, or both, rather than the result of unmasking of sulphated glycosaminoglycans.     

Ultrastructural histochemistry of the surface lamina of normal articular cartilage

Summary Two collagen-poor, ultramicroscopic layers are described at the surface of canine articular cartilage. They are distinguished by staining with an electron-dense cationic dye, Cupromeronic Blue, in a critical electrolyte concentration technique and by digestion with testicular hyaluronidase. The superficial layer, approximately 50 nm thick, stained at low electrolyte concentrations but failed to stain in conditions specific for sulphated glycosaminoglycans. It was hyaluronidase-resistant and may be either glycoprotein or protein in nature. The deeper layer, 100–400 nm thick, stained positively at electrolyte concentrations specific for sulphated glycosaminoglycans but not in conditions specific for keratan sulphate. It was removed by hyaluronidase digestion. This layer probably represents a chondroitin sulphate-rich proteoglycan.These surface layers may be important in the lubrication of the articular surface and in the permeability and compression resistance of the superficial cartilage zone.     

Electron microscopic observations on pulmonary connective tissue stained by Ruthenium Red

Summary Ultrastructural studies on human lung were performed with special attention to the interstitial acid mucopolysaccharides by Ruthenium Red staining and several enzyme digetion tests withStreptomyces hyaluronidase, chondroitinase ABC, chondroitinase AC, heparinase, trypsin and collagenase.Periodic lateral granules on the major cross bands of collagen fibrils and amorphous coats on them became visible by Ruthenium Red staining. The surface of elastic fibres, associated microfibrils, and some fine fibrils 10–20 nm in diameter were stained. Ruthenium Red also stained the surface of fibroblast and smooth muscle cells, basement membrane and filamentous long segments. In the interstructural space, granular substances 10–80 nm in diameter and fine filaments 3–4 nm thick, which formed a fine reticular network, were clearly observed. They were not visible on the usual thin section. The granular substances were located on the cross points of the fine filaments. They spread continuously and connected with each of the cells and extracellular structures in the pulmonary interstitium. The results of the enzyme digestion tests on the Ruthenium Red-positive material are discussed.     

Fixation and embedding variables in the immuno-electron microscopic study of rat heymann nephritis

Summary Fixation and embedding variables were compared in immuno-electron microscopic localization of rat IgG in an autologous immune complextype nephritis. Specimens from kidney cortex were fixed for 3, 6 or 9 h in the following fixatives made in 0.1 M phosphate buffer at pH 7.44% paraformaldehyde, 2.5% glutaraldehyde, periodate-lysine-paraformaldehyde or modified Karnovsky's fixative. Localization of IgG was performed on tissue sections cut with a tissue chopper, cryostat or sliding microtome, using agarose, Ames O.C.T. Compound or polyethylene glycol respectively as cutting matrixes. The sections were incubated in peroxidase-labelled antirat IgG antiserum (diluted 120 with phosphate-buffered saline) for 60 h. Peroxidase activity was then revealed and the sections embedded in Epon. Exact localization of IgG throughout the sections and good ultrastructure were achieved when paraformaldehyde and agarose were used. Periodate-lysine-paraformaldehyde proved almost as useful as paraformaldehyde in connection with agarose in respect of peroxidase reaction and ultrastructure. Fixatives containing glutaraldehyde gave a mostly weak and unevenly distributed peroxidase reaction product. In the cryostat sections breaking of the tissue structure could not be avoided. When polyethylene glycol was used as cutting matrix no peroxidase reaction was achieved.     

The tidemark of the chondro-osseous junction of the normal human knee joint

Summary The chondro-osseous junction includes the junction between calcified and non-calcified cartilage matrices often referred to as the tidemark. A detailed knowledge of the structure, function and pathophysiology of the chondro-osseous junction is essential for an understanding both of the normal elongation of bones and of the pathogenesis of osteoarthrosis. In this study the molecular anatomy of the tidemark was studied using histochemical techniques, including lectin histochemistry, on blocks of normal cartilage from human knee joints. The tidemark stained with H&E, picro-sirius red, toluidine blue, safranin O and methyl green, but not with alcian blue in the presence of magnesium chloride at 0.05 M or above. It stained with only four lectins, those from Datura stramonium, Maclura pomifera, Erythrina crystagalli and Helix pomatia, out of the 19 used. Therefore, it is rich in collagen and contains hyaluronan, but appears to lack the glycosaminoglycans of conventional proteoglycans and it expresses a very limited and distinctive lectin staining glycoprofile, which is probably attributable to specific glycoproteins. In addition, the tidemark had a distinct microanatomical trilaminate appearance. From all of these results it is clear that this part of the chondro-osseous junctional region is chemically more complex and distinctive than has previously been described.