Bx fluorescence microscope

Biopsied renal tissue was placed in Zeus fixative (Zeus Scientific, Branchburg, NJ), sectioned using a cryostat, and then stained as follows in a multistep procedure. Initially, the Zeus-fixed tissue was placed in Zeus fixative wash solution (Zeus Scientific) and sectioned in a cryostat at 4 μm thickness, and the resultant sections were placed on poly-L-lysine-coated slides at room temperature, allowed to dry, and then fixed in cold acetone for 10 minutes. The tissue sections were then washed in PBS times 3 for 10 minutes each. The sections were then stained using a 3-step procedure as follows: (1) mouse anti-human C4d (Quidel Corp., San Diego, CA) was diluted 1 : 200, for 30 minutes and then washed in PBS; (2) FITC-conjugated rabbit anti-mouse immunoglobulin (Dako Corp, Carpinteria, CA) diluted 1 : 30, for 20 minutes, with interval wash in PBS; and (3) finally FITC-conjugated swine anti-rabbit immunoglobulin (Dako Corp), diluted 1 : 30, for 20 minutes, with final wash in PBS. Slides were then mounted with a cover slip in glycerol/Optimax solution and reviewed in an Olympus BX Fluorescence Microscope.