Dp70 camera and software

Anesthetized mice were perfused through the left ventricle of the heart with phosphate-buffered saline, followed by Bouin’s fixative. Thyroid glands and testes were dissected out of the body, fixed in Bouin’s for 7 days, and paraffin embedded. Inner ears were held in Bouin’s fixative for 4 weeks before embedding. For eye preparations, mice were asphyxiated by carbon dioxide inhalation without perfusion. Enucleated eyes were fixed overnight in cold methanol/acetic acid solution (3:1, v/v), and paraffin embedded. All sections were mounted on glass slides and counterstained for microscopic examination. Sections of thyroid glands (6 μm), eyes (6 μm), and inner ears (4 μm) were stained with H&E. Sections of testes (5 μm) were stained with Periodic acid–Schiff (PAS) stain. All slides were examined on an Olympus Optical BX40 light microscope (Olympus, Tokyo, Japan), and images were captured and processed utilizing the Olympus DP70 camera and software.

Formalin-fixed, paraffin-embedded kidneys were cut into 5 μm sections. Slides were deparaffinized, rehydrated from xylene through a graded ethanol series to ddH₂O and subsequently treated as described below. Slides were viewed on a Nikon EclipseE600 microscope (Melville, NY) and analyzed with an Olympus DP70 camera and software or an Olympus BX51 microscope equipped with an Olympus DP70 camera and software (Olympus America Inc., Center Valley, PA). All H&E and C4d slides were reviewed by Dr. Weixiong Zhong, MD, PhD, transplant pathologist, and scored for ptc, glomerulitis (g), vasculitis (v)/intimal arteritis, interstitial inflammation (i) and C4d staining, according to Banff 2009 (29 (link),30 (link)). Morphometric analysis using electron microscopy was performed using standard methodology, as described earlier by the UW Department of Pathology and Laboratory Medicine (31 (link)).