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

Abstract

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.