Alterations in stromal glycoconjugates in macular corneal dystrophy

Nine biotinylated lectins were used as histochemical probes to localize the carbohydrates residues of glycoconjugates in normal corneas and in corneas with macular and granular dystrophy. The lectin binding patterns of normal corneas and of corneas with granular dystrophy were indistinguishable from one another, but were distinctly different from those found in corneas with macular dystrophy. Concanavalin A reacted weakly with normal corneal stromal matrix, but stained stromal matrix of corneas with macular dystrophy intensely. Furthermore, unlike the normal corneal matrix, stromal matrix of corneas with macular dystrophy reacted positively with wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA-I), Ulex europeus I, Dolichos biflorus, Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, and soybean and peanut lectins. This study demonstrates specific alterations in glycoconjugates which occur in the corneal matrix of patients with macular dystrophy, namely the presence of oligosaccharides with terminal alpha-fucose, beta-galactose, N-acetylglucosamine and N-acetylgalactosamine residues, and oligosaccharide chains with a beta-galactose-N-acetylgalactosamine sequence.     

Altered lectin binding sites in keratoconus corneas.

We investigated the glycoconjugates in frozen sections of keratoconus corneas, using a panel of 12 biotin- or fluorescein isothiocyanate-labeled lectins. No differences between the lectin binding sites of the epithelium, endothelium and Descemet's membrane of normal and keratoconus corneas could be observed. However, in contrast to normal corneas, intense staining with peanut agglutinin (PNA) could be detected at breaks in Bowman's layer, in scar tissue and in the adjacent stroma. Furthermore, in the majority of cases binding sites for Phaseolus vulgaris erythroagglutinin (PHA-E) and increased staining with Ricinus communis agglutinin I (RCA-I) and Lens culinaris agglutinin (LCA) could also be detected in ruptures in Bowman's layer and in scar tissue. These data suggest that the scarred regions of the anterior stroma in keratoconus corneas may contain oligosaccharides with terminal D-galactose (beta 1-3)-D-N-acetylgalactosamine disaccharides (recognized by PNA), increased amounts of glycoconjugates with terminal beta-galactose residues (recognized by RCA-I), increased amounts of glycoconjugates with glucose/mannose residues (recognized by LCA), and finally, biantennary complex-type glycopeptides containing two outer galactose residues and a residue of N-acetylglucosamine (recognized by PHA-E). Since corneal scars due to causes other than keratoconus revealed lectin binding sites (particularly for PNA and to a lesser extent also for PHA-E, LCA and RCA-I) similar to those seen in scar tissue of keratoconus corneas, we conclude that it is mainly scar formation that may be responsible for the altered lectin binding sites in keratoconus.     

Abnormal extracellular matrix in corneas with pseudophakic bullous keratopathy

The present study describes biochemical and morphological differences of pseudophakic bullous keratopathy (PBK) corneas as compared with normal corneas. At the ultrastructural level, all PBK corneas studied had abnormal fibrillar material posterior to the Descemet's membrane. In addition, two of the six PBK buttons had subepithelial fibrocellular materials disrupting the epithelial basement membrane and Bowman's layer. Aggregates of collagen fibrils with 110 nm periodicity were occasionally seen within the stroma of the PBK corneas. Isolation and purification of the collagen from the Descemet's membrane/posterior collagenous layer (DM/PCL) showed an increased amount of material with molecular weight in the range of 50-60K daltons (presumably type VIII collagen) and decreased amounts of higher molecular weight, disulfide-bonded collagenous materials (presumably type IV collagen) as compared with normals. Sugar-specific lectin studies showed an increased deposition of peanut agglutinin (PNA) and Ricinus communis agglutinin I (RCA120) in the DM/PCL of the PBK corneas. Our data suggest that the DM/PCL of PBK corneas have an increased accumulation of terminal B-galactose and B-D-galactose (1-3)-D-N-acetylgalactosamine residues and altered ratios of low and high molecular weight collagenous proteins.     

人正常发育角膜上皮和角膜基质的凝集素化学研究

目的:研究正常人发育角膜上皮及基质中凝集素亲和糖蛋白的分布及变化,探讨糖类化合物对角膜发育的可能作用。方法:用五种生物素标记的凝集素:麦胚凝集素(Wheat Germ Agglutin WGA)、伴刀豆凝集素(Con-Canavlin A ConA)、花生凝集素(Peanut Agglutin PNA)、蓖麻凝集素(Ricinus Communis Agglutin I RCA-I)扁豆凝集素(Lens Culinaris A LCA)对人正常发育角膜进行凝集素组织化学研究。结果:1.WGA受体主要分布在角膜上皮细胞膜中,尤其在表层上皮细胞中有明显分布,在发育期间胚胎角膜的前部基质中也有WGA受体分布。2.ConA受体分布于角膜上皮细胞膜及细胞质内,在胚胎期角膜基质中有不均一分布。3.RCALCA受体主要分布在角膜上皮基底部细胞膜上。4.PNA受体在胚胎16周角膜上皮细胞中有较少量分布。结论:1.在人正常发育角膜组织中含有ConA、WGA、PNA、LCA、RCA亲和性糖蛋白,这些糖蛋白参与发育角膜构成。2.在人角膜发育成熟过程中,有凝集素亲和糖蛋白的分布变化,这种变化可能与角膜的发育成熟有关。眼科学报1996;12:178～182。     

Keratoconus: I. Biochemical studies

The present study analyses collagenous and non-collagenous components from age-matched normal and keratoconus corneas. Intact keratoconus corneas showed decreased collagen, total protein, and hydroxylysine levels, with normal reducible collagen cross-linking. Non-collagenous fractions were isolated from corneas with a 4 M guanidine procedure. As demonstrated by PAGE-silver stain, the keratoconus cornea guanidine extracts had a 75 kDa band that was absent in normal cornea guanidine extracts. In addition, there was a markedly increased level of protein, uronic acid and neutral hexose in keratoconus extracts as compared with controls. Our Western blot studies showed increased affinity for the castor-bean agglutinin (RCA120, specific for terminal galactose) in the keratoconus extracts as compared with normals. These data suggest the presence of an abnormal noncollagenous component in keratoconus corneas.     

Extracellular matrix and Na+,K+-ATPase in human corneas following cataract surgery: comparison with bullous keratopathy and Fuchs' dystrophy corneas.

PURPOSE: To examine the distribution of extracellular matrix (ECM) and basement membrane (BM) components and of Na+,K+-ATPase in postcataract surgery (PCS) corneas. These corneas were from patients who never developed pseudophakic or aphakic bullous keratopathy (PBK/ABK) after cataract surgery. PCS corneas were compared with PBK/ABK and Fuchs' dystrophy corneas. METHODS: The distribution of PBKIABK ECM and BM markers and of all three Na+,K+-ATPase alpha subunits was studied by immunofluorescence in 10 healthy, 11 PCS, 16 PBK/ABK, and 12 Fuchs' dystrophy corneas. RESULTS: Fibrotic ECM proteins, tenascin-C and fibrillin-1, were found in only 1 of 10 healthy and in 2 of 11 PCS corneas. In contrast, these proteins were expressed in all PBK/ABK and more than half of the Fuchs' dystrophy corneas. BM components in PCS corneas were altered to a greater extent (40-60%), especially fibronectin and laminin-10. A decreased epithelial immunostaining for Na+,K+-ATPase alpha subunits was seen in approximately 40% of PCS corneas and in approximately two thirds of PBK/ABK and Fuchs' dystrophy corneas. However, the endothelial staining was normal in all groups. CONCLUSIONS: Because tenascin-C and fibrillin-1 were mostly found in diseased but not in PCS corneas, their expression may be related to later, clinical stages of corneal edema development. However, BM components abnormal in PBK/ABK and Fuchs' dystrophy corneas were also altered in PCS corneas without clinical evidence of ocular disease. This may result from subclinical corneal changes resulting from cataract surgery, lens removal, exposure to the intraocular lens, or endothelial cell damage. Alterations of epithelial Na+,K+-ATPase point to the importance of epithelial changes in the development of corneal edematous diseases.     

Two-dimensional gel electrophoretic comparison of endothelial cell-Descemet's membrane proteins in Fuchs' dystrophy and normal corneas.

Two-dimensional polyacrylamide gel electrophoresis was used to compare the proteins isolated from the combined corneal endothelial cell-Descemet's membrane complex of three pairs of corneas with Fuchs' dystrophy with three pairs of normal corneas. Normal or Fuchs' dystrophy endothelium and Descemet's membrane was documented by pathologic analysis of each cornea. Proteins were separated by isoelectric point in the first dimension and molecular weight in the second dimension. Over 300 proteins were resolved from each sample, and similar patterns were noted in both groups. No consistent differences were detected between the corneas with Fuchs' dystrophy and the normal corneas. Allelic variations of some proteins were detected in both groups.     

Epithelial cell, keratocyte, and endothelial cell apoptosis in Fuchs' dystrophy and in pseudophakic bullous keratopathy

PURPOSE: To elucidate the pathomechanism of Fuchs' dystrophy and pseudophakic bullous keratopathy (PBK) by examining cell apoptosis in different corneal layers. METHODS: The authors studied corneal buttons obtained from 21 eyes following central penetrating keratoplasty: 14 corneal buttons (13 patients, age 70.8+/-10.0 years) with Fuchs' dystrophy, and 7 buttons (7 patients, age 69.6+/-10.2 years) with PBK. Four buttons from enucleated eyes with choroidal melanoma served as controls. Histologic changes were examined using light microscopy with hematoxylin-eosin (HE) staining. The average numbers of apoptotic cells per field of view (125x magnification) in separate samples of the epithelial, stromal, and endothelial layers were determined using the TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling) assay. RESULTS: In 11 of the Fuchs' dystrophy corneas and 2 of the PBK corneas, apoptotic activity was detected. In the control corneas no apoptotic activity was found. Compared to the controls there was a statistically significant difference in the mean (normalized) apoptotic cell numbers for all three layers (p=0.01 in each case) in the Fuchs' dystrophy corneas, and for the stromal layer (p<0.01) in PBK corneas. The apoptotic cell numbers for the epithelial and endothelial layers of the latter were higher, but the difference was not statistically significant (p=0.07, 0.07). CONCLUSIONS: Apoptosis may play a role in the pathomechanism of Fuchs' dystrophy and in keratocyte death in corneas with PBK.     

CD-34 stromal expression pattern in normal and altered human corneas

OBJECTIVE: To test CD-34 immunoreactivity in stromal cornea cells in normal and pathologic samples obtained from penetrating keratoplasty. DESIGN: Prospective, consecutive histopathologic human tissue study. PARTICIPANTS AND CONTROLS: One hundred two cornea buttons from patients with different diseases, submitted for cornea transplant, were examined. Controls were expired corneas from healthy donor patients who died (n = 4), and globes enucleated for primitive intraocular neoplasias, that is, retinoblastomas (n = 8), and malignant choroidal melanomas (n = 2). METHODS: The expression of CD-34 in stromal cornea cells was examined by immunohistochemistry analysis. Other immunohistochemical stains included an endothelial cell marker (CD-31), common leukocyte antigen, and alpha-smooth muscle actin. MAIN OUTCOME MEASURES: Different diseases that may cause blindness and require penetrating keratoplasty have been tested for CD-34 immunoreactivity. RESULTS: In control corneas, keratocytes present strong and consistent CD-34 immunoreactivity. Diseases leading to the loss of transparency and penetrating keratoplasty, such as keratoconus, herpes keratitis, trauma, and heredofamilial dystrophies, are associated with focal or diffuse loss of CD-34 expression, whereas pseudophakic bullous keratopathy and Fuchs' endothelial dystrophy show normal CD-34 immunoreactivity in most cases and patchy unstained stromal areas in a few cases. CONCLUSIONS: Scar tissue formation in the cornea, as in herpes keratitis and trauma, is always associated with loss of CD-34 immunoreactivity, which may otherwise be a primary event in keratoconus and heredofamilial dystrophies. Both in the pseudophakic bullous keratopathy and Fuchs' endothelial dystrophy, CD-34 immunoreactivity may be normal or lost, hence these two diseases may be considered as one and part of the same group with regard to CD-34 expression, as revealed by immunohistochemistry analysis.     

Cytochrome oxidase activity of postsurgical bullous keratopathy endothelium

Proper corneal hydration is normally maintained by a pump-leak mechanism located on the lateral cell membranes of the endothelium (1,2). When endothelial cells become sufficiently dysfunctional in response to trauma, the cornea becomes irreversibly edematous and opaque. Previous studies in our laboratory have shown that mitochondrial cytochrome oxidase (CO) activity, an enzyme important in respiratory activities, is correlated with endothelial cell functional activity in corneas with Fuchs' endothelial dystrophy. In this study we investigated cytochrome oxidase activity in corneas with postsurgical bullous keratopathy. Corneas with aphakic and pseudophakic bullous keratopathy (ABK, PBK) were incubated in diaminobenzidine-cytochrome C media. Results showed that the staining pattern of ABK and PBK corneas was uniformly low compared to keratoconus corneas, and in contrast to previous studies on Fuchs' dystrophy corneas which demonstrated a regional staining pattern. This suggests that CO staining patterns correlate with functional activity and may be a useful technique to detect dysfunctional cells in various disease categories.     

Characterization of the Descemet's membrane/posterior collagenous layer isolated from Fuchs' endothelial dystrophy corneas

The combined Descemet's membrane (DM) and posterior collagenous layer (PCL) of Fuchs' endothelial dystrophy corneas were isolated and characterized by biochemical and immunofluorescence methods. The amino acid composition of the Fuchs' DM-PCL was similar to age-matched normal Descemet's membranes (DM). As determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ¹²⁵I two-dimensional peptide mapping, normal DM and Fuchs' DM-PCL contained the same collagen types [type IV and endothelial cell (EC) collagen], but a slight discrepancy was seen in the electrophoretic mobility of some collagen chains. Immunofluorescence staining localized fibrinogen/fibrin to Fuchs' DM-PCL but not to normal DM. These data suggest (1) that the appearance of 110 nm banded material in sheets and fusiform bundles characteristic of Fuchs' PCL is not due to the presence of a new (abnormal) collagen type but may represent altered assembly of collagen molecules, and (2) that the fibrinolytic system may play a role in the degenerative process of Fuchs' endothelial dystrophy.     

Blood staining of the cornea. Light microscopic and ultrastructural features

Eleven blood-stained corneas were examined by light and transmission electron microscopy at intervals ranging from one month to seven years after initial staining occurred. Blood staining in each case was associated with focal loss of endothelial cells or endothelial degenerative changes and elevated intraocular pressure. Globules of erythrocytic breakdown products penetrated the discontinuous endothelium and intact Descemet's membrane. Large deposits, primarily extracellular, displaced but did not interrupt the stromal lamellae. Keratocytes in blood-stained corneas contained erythrocytic breakdown products and hemosiderin, and were remarkable for extensive degenerative changes in contrast to keratocytes in areas of cleared cornea, which contained smaller amounts of hemosiderin and were relatively normal. After one year, clearing could be seen to involve peripheral and posterior stroma, and to a lesser degree, the anterior stroma. We found no evidence to support the contention that blood-derived macrophages play a role in the clearing of erythrocyte debris. The stereotyped pattern of peripheral, posterior, and anterior stromal clearing observed seems to be consistent with diffusion of hemoglobin breakdown products out of the cornea as the primary mechanism of clearing.     

Growth of human corneal endothelium on altered Descemet's membrane.

To determine whether Descemet's membrane (DM), which is altered by disease, interferes with endothelial cell growth, healthy human corneal endothelial cells were seeded onto DM from normal corneas and diseased corneal buttons from patients with Fuchs' endothelial dystrophy and pseudophakic bullous keratopathy (PBK). DM was first peeled off the corneal specimens and the endothelial cells removed by trypsinization. A suspension of first-passage corneal endothelial cells (2,000 cells/microliters; obtained from donor eye bank eyes and grown in Dulbecco's minimal essential medium with 10% fetal bovine serum and 1.5% chondroitin sulfate) were seeded on DM. Epidermal growth factor (10 ng/ml) and insulin (1 U/ml) were added to the medium after seeding cells on the DM. The cells attached and flattened within 1 hour and reached confluency in 1 week on normal DM. Cells grown on DM from corneas of patients with Fuchs' endothelial dystrophy also flattened and grew to confluency in 1 week. Cells grown on DM from corneas of patients with PBK did not grow to confluency. Further studies with bovine and rabbit corneal endothelial cells showed similar growth pattern to human cells. These data indicate that DM from corneas of patients with Fuchs' dystrophy does not interfere with the growth of corneal endothelial cells but that DM from corneas of patients with PBK does interfere with cell growth.     

A comparison of corneal endothelial morphology in cornea guttata, Fuchs' dystrophy and bullous keratopathy

Changes on corneal endothelial specular microscopy were compared in 14 patients with cornea guttata, 4 patients with Fuchs' corneal dystrophy and 19 patients with various forms of bullous keratopathy. The patients with cornea guttata showed well marked guttae 1 to 6 endothelial cells in diameter in the endothelial mosaic and in the relief mode while the endothelial mosaic was usually otherwise within normal limits. In 2 patients with Fuchs' dystrophy the endothelium could be examined, showing gross guttae but a few areas of relatively normal endothelial cells. The unaffected eye of 3 other patients showed findings similar to cornea guttata, but with some reduction in endothelial cell count in 2 patients. The patients with bullous keratopathy fell into 2 groups, one with gross reduction in cell count in a markedly abnormal endothelial cell mosaic, the other a mixed group with moderate reduction in cell count and numerous guttae. Some miscellaneous cases included one of aphakic peripheral bullous keratopathy, one associated with cyclitis and aphakia and 2 with idiopathic non-surgical bullous keratopathy. We believe the corneal endothelium is not grossly abnormal away from the guttae in Fuchs' dystrophy, but the gross guttata formation determines the endothelial dysfunction.     

A COMPARISON OF CORNEAL ENDOTHELIAL MORPHOLOGY IN CORNEA GUTTATA, FUCHS' DYSTROPHY AND BULLOUS KERATOPATHY

Changes on corneal endothelial specular microscopy were compared in 14 patients with cornea guttata, 4 patients with Fuchs' corneal dystrophy and 19 patients with various forms of bullous keratopathy. The patients with cornea guttata showed well marked guttae 1 to 6 endothelial cells in diameter in the endothelial mosaic and in the relief mode while the endothelial mosaic was usually otherwise within normal limits. In 2 patients with Fuchs' dystrophy the endothelium could be examined, showing gross guttae but a few areas of relatively normal endothelial cells. The unaffected eye of 3 other patients snowed findings similar to cornea guttata, but with some reduction in endothelial cell count in 2 patients. The patients with bullous keratopathy fell into 2 groups, one with gross reduction in cell count in a markedly abnormal endothelial cell mosaic, the other a mixed group with moderate reduction in cell count and numerous guttae. Some miscellaneous cases included one of aphakic peripheral bullous keratopathy, one associated with cyclitis and aphakia and 2 with idiopathic non-surgical bullous keratopathy. We believe the corneal endothelium is not grossly abnormal away from the guttae in Fuchs' dystrophy, but the gross guttata formation determines the endothelial dysfunction.     

Fuchs corneal dystrophy: aberrant collagen distribution in an L450W mutant of the COL8A2 gene

PURPOSE: To characterize histologically Descemet's membrane in an early-onset Fuchs corneal dystrophy (FCD) COL8A2 mutant and compare these findings with corneas from late-onset FCD and normal corneas. METHODS: A corneal explant from a patient with the L450W COL8A2 mutation and others with late-onset disease were studied with antibodies to collagens IV, VIIIA1, VIIIA2, fibronectin, and laminin. Transmission electron microscopy was performed on a portion of the explant. Control explants included eye bank corneas without known disease and surgical explants from unrelated conditions. RESULTS: In normal corneas, a regular array of colocalized COL8A1 and COL8A2 was observed in the anterior half of Descemet's membrane. In the L450W mutant, Descemet's membrane was several times thicker than normal and traversed by refractile strands and blebs that stained intensely for COL8A2, a feature also observed in late-onset FCD. Both the alpha1 and alpha2 subtypes of collagen VIII were observed at high levels along the anterior edge of Descemet's, another abnormal feature also found in late-onset FCD. Ultrastructure of the L450W cornea revealed a well-formed anterior banded layer more than three times thicker than normal. An unusual, thin internal layer was rich in patches of wide-spaced collagen. The layer is a distinctive pathologic structure that is associated with FCD and is characterized by approximately 120 nm periodicity and the presence of collagen VIII. Depositions of collagen IV, fibronectin, and laminin were also greatly increased in the of posterior Descemet's membrane, yet another general feature shared between early- and late-onset disease. CONCLUSIONS: Early-onset COL8A2 L450W disease involves massive accumulation and abnormal assembly of collagen VIII within Descemet's membrane, a process that is presumed to begin during fetal development. Both early- and late-onset subtypes of FCD appear to be the result of abnormal basement membrane assembly rather than a primary defect in endothelial metabolism.     

Lectin receptors in the human cornea

Five different biotin labeled lectins, Concanavalin-A (Con A), wheat germ agglutinin (WGA), Ricinus communis agglutinin I (RCA1), Ulex europaeus agglutinin (UEA1), and soybean agglutinin (SBA) were used to study lectin receptors on formalin-fixed paraffin embedded human corneas. Con A stained the cytoplasm, cell, and nuclear membranes of the epithelial cells and stained the stroma diffusely. WGA stained the superficial epithelial cells, the epithelial cell membranes, and the keratocytes of the stroma. SBA did not react with any of the corneal layers. RCA1 heavily stained the keratocytes but did not stain the epithelium. UEA1 lightly stained the epithelial cell cytoplasm and interstitial stroma. All staining reactions could be abolished by omission of the lectin or by the use of the appropriate inhibitory sugar. The lectin binding patterns reported here provide a means for further investigation of carbohydrate structures in the human cornea in both normal and disease states.     

Cultured trabecular-meshwork cells: immunohistochemical and lectin-binding characteristics

We examined the extracellular matrix formation and lectin-binding properties of cultured trabecular-meshwork cells established from cynomolgus monkey and bovine eyes. Using an avidin-biotin complex method, we found that the extracellular matrix in both the monkey and bovine cultures stained intensely with antibodies to fibronectin, type IV collagen, and laminin. These materials were especially prominent when the cultured monkey trabecular-meshwork cells aggregated in clusters, manifesting their close anatomic relationship to trabecular beams. Cell-surface and intracellular lectin-binding properties were studied using eight biotinylated lectins: concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), Dolichos biflorus agglutinin (DBA), soy bean agglutinin (SBA), Phaseolus vulgaris (PHA), wheat germ agglutinin (WGA), and Ulex europaeus agglutinin I (UEA I). Both monkey and bovine trabecular-meshwork cells showed positive cell surface and intracellular binding to Con A, WGA, and PHA. Only moderate cell-surface staining was observed with RCA, and no visible staining occurred with PNA, DBA, SBA, and UEA-1.     

Distribution of specific collagen types and fibronectin in normal and keratoconus corneas

The distribution of five types of collagen and fibronectin in 6 normal and 9 keratoconus corneas was examined, using immunofluorescent staining and the enzyme-labeled antibody method. Types I, III and V collagens were detected in the corneal stroma. There was essentially no difference between normal and keratoconus corneas in their distribution. Type IV collagen and fibronectin were detected in the basement membrane of the normal corneal epithelium, while in the keratoconus corneas the disruption of the basement membrane as well as the excrescence of basement membrane materials was observed. The abnormal distribution of the type IV collagen and fibronectin was also observed in the anterior stromal area of keratoconus corneas.     

New method of ultrastructural localization of WGA-binding sites in the rabbit cornea using quick-frozen specimens followed by freeze-substitution

Wheat germ agglutinin (WGA) conjugated with apo-horseradish peroxidase-gold was used as a probe for ultrastructural localization of N-acetyl-glucosamine and N-acetyl-neuraminic acid residues of glycoconjugates in posterior stroma and Descemet's membrane of rabbit corneas. Ultra-thin sections of quick-frozen, freeze-substituted, and Epon or Lowicryl K4M-embedded tissue were used. Numerous WGA-binding sites were detected in the stromal matrix and Descemet's membrane in both embedding procedures. Labelling was more intense with low-temperature embedding in Lowicryl K4M than with Epon. Gold particles preferentially labelled the major collagen fibrils of the stroma but otherwise appeared uniformly distributed over both extracellular matrices, except in some areas of the stromal matrix that were barely labelled. Frequently, the inter-particle distance of gold particles on collagen fibrils corresponded to the banding period of type I collagen fibrils, suggesting that they point out keratan sulphate proteoglycans which contain WGA-binding sites. The combination of cryotechniques and post-embedding procedures may be very effective for lectin histochemistry of the cornea and other eye tissues.