Tissue preparation for immunohistochemistry.

Mice were anesthetized with inhalant isoflurane. The kidneys were preserved by in vivo cardiac perfusion with PBS (pH 7.4) containing 6,000 unit/l of Na-heparin and 120 mg/l lidocaine followed by periodate-lysine-2% paraformaldehyde (PLP) and then cut transversely into several 2- to 3-mm-thick slices and immersed for 24–30 h at 4°C in the same fixative. Kidney samples from each animal were embedded in polyester wax using polyethylene glycol 400 distearate (Polysciences, Warrington, PA) with 10% 1-hexadecanol, and 2-μm-thick sections were cut and mounted on gelatin-coated glass slides.