A comparison of different embedding media on the ultrastructure of the trabecular meshwork

Purpose. To determine whether the preservation of the extracellular matrix and the ultrastructural appearance of the trabecular meshwork are affected by different histologic processing protocols and embedding media. Conventio nally used epoxy resins such as Araldite require complete dehydration of ti ssue, while the acrylic resin LR White requires only partial dehydration, b etter preserves tissue antigens, and has been reported to preserve more of the extracellular matrix. Methods. Seven human eyes ranging in age from 2 months to 78 years were dis sected and tissue samples from each eye processed and embedded in both Aral dite and LR White media. The ultrastructure of the trabecular cells and the extracellular matrix of the meshwork was compared between media. The prese rvation of the extracellular matrix in the juxtacanalicular region was dete rmined by measuring the amount of material immediately underlying Schlemm's canal. Immunoelectron microscopy was used to determine the composition of this material. Results. Araldite provided better resolution of ultrastructural details tha n freshly polymerized LR White. After a period of ripening for several mont hs, however, resolution of tissue details in LR White improved. No signific ant quantitative difference was found in the amount of extracellular matrix underlying Schlemm's canal when comparing the two media. Neither post-mort em time to fixation (up to 31 hr), donor age, nor immersion vs, perfusion f ixation technique affected the amount of extracellular material present in the comparison of the two embedding media. Immunogold labeling of the extra cellular material within the juxtacanalicular tissue revealed the presence of collagen IV, laminin, and fibronectin in the basement membrane region im mediately underlying the inner wall, and also in scattered patches within t he juxtacanalicular tissue. Conclusion. Despite the less rigorous processing required for LR White than epoxy embedding, neither the appearance nor amount of the extracellular ma terial was affected by the different embedding protocols. Prolonged post-mo rtem time to fixation did not affect the appearance nor amount of extracell ular matrix. Immunolabeling revealed that the extracellular matrix of the j uxtacanalicular tissue contains components of basement membrane material.