Fifteen normally pigmented golden hamsters (Mesocricetus auratus) were used for this study. Animals were anesthetized by intraperitoneal injection of 0.85 mg pentobarbital sodium (Nembutal; Abbott Laboratories, Abbott Park, IL) per gram of body weight in accordance with the ARVO Statement and guidelines established by the University of Southern California and St. Louis University Animal Care Committees. Hamster and human tissues used for freeze-fracture were obtained after perfusion or immersion fixation in 2% formaldehyde (generated fresh from paraformaldehyde) and 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), containing 0.025% calcium chloride. Tissues were stored at 4°C in 0.1 M sodium cacodylate buffer. They were later cut into 100- to 150-μm thick sections with a tissue chopper (TC-2; Sorvall, Newtown, CT). Tissues were passed through solutions of 10%, 20%, and 30% glycerol and 0.1 M cacodylate buffer, over 60 to 90 minutes, and then placed between gold alloy specimen discs and frozen immediately in Freon-22 (DuPont, Wilmington, DE). Discs were placed in a double-replica specimen holder that was precooled in liquid nitrogen and subsequently transferred to a freeze-fracture unit (model 301; Balzers, Hudson, NH).
Fracturing, shadowing, and replication were accomplished at a vacuum of at least 10−6 mm Hg and at a specimen post temperature of −119°C. The postreplication temperature was raised to −20°C in some cases before the second coat of carbon was evaporated over the fractured specimen. Replicas were prepared with minimum etching and subsequently cleaned in 100% methanol for 1 hour, 5% sodium hypochlorite for 12 to 24 hours, 50% sulfuric acid for 1 to 4 hours, and three to four rinses of distilled water. Replicas were picked up on resin-coated copper grids (Parlodion; SPI Supplies, West Chester, PA) and observed in a transmission electron microscopes equipped with goniometer stages (models 100C or 1200EX; JEOL).