We studied 13 human donor eyes (13 donors) with ages varying between 21 and 80 years (median donor age was 63 years). Eyes were obtained from the Cornea Bank (Amsterdam, The Netherlands) after removal of the corneas for transplantation. The provisions of the Declaration of Helsinki for research involving human tissue were observed. Specimens were studied by LM and TEM. Specimens were fixed by immersion within 48 hours after death in 2% glutaraldehyde (GA, TAAB Laboratories, Aldermastron, UK) in 0.1 M phosphate buffer (pH 7.4) for several days. After removal of small parts of the globe at 6 and 12 o’clock, specimens were fixed for an additional 4 hours in the same fixative. Specimens were washed in 6.8% sucrose in phosphate-buffered saline (PBS) for 2 hours and briefly in double-distilled water. After transection of the globes, specimens were immersed for 20 hours in 0.2% aqueous cupromeronic blue (CB; Seikagaku Co., Tokyo, Japan) containing 0.025 M NaAc, 0.3 M MgCl2, and 2.5% GA. Specimens were briefly washed in staining solution minus CB, washed in double-distilled water, and either dehydrated through ethanol-propylene oxide mixtures and embedded in Epon 812 (Serva Feinbiochemica, Heidelberg, Germany) or dehydrated through increasing concentrations of ethanol and embedded in GMA (Technovit 7100; Heraeus Kulzer). The GMA-embedded materials were cut on a microtome (Reichert Jung, Vienna, Austria). Sections 3 to 4 μm thick were stained with toluidine blue (TB) and evaluated by LM. Areas of interest were selected for evaluation by TEM and cut from larger plastic blocks. Small plastic blocks were cut on a second microtome (Sorvall, Newtown, CT). Thin sections approximately 200 nm thick were mounted on polyvinyl formal–coated grids (Formvar; SPI, West Chester, PA), counterstained with uranyl acetate in 25 centipoise methylcellulose and evaluated with a TEM (model 201; Philips, Eindhoven, The Netherlands) operated at 80 kV. For the Epon-embedded materials, the areas of interest were directly selected for evaluation by TEM and cut from the larger plastic blocks. Semithin sections (1–3 μm) were stained with TB and evaluated by LM. Ultrathin sections (80–90 nm) were contrasted with uranyl acetate and lead citrate and examined with the TEM operated at 60 kV.
Chondroitin sulfate proteoglycan (CS-PG) is the predominant PG in the vitreous.
20 21 It can be removed by the action of chondroitinase ABC. Parts of some specimens (
n = 5) were subjected to chondroitinase ABC treatment before CB staining as follows. Specimens were incubated overnight at 37°C in chondroitinase ABC 0.1 U/mL (C-3667; Sigma-Aldrich, St. Louis, MO) in 25 mM Tris-HCl (pH 8.0) containing 2 mM MgCl
2, washed in Tris buffer and in double-distilled water.