Mouse Kidney Tissue Preparation and Immunohistochemistry

Animal experiments were approved by the Institutional Committee on Research Animal Care, following the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult C57BL/6J mice received a single intraperitoneal injection of colchicine (0.5 mg/100 g body wt) or saline. After 2 h, mice were anesthetized by 2% isoflurane inhalation. The kidneys were perfused with PBS (37°C) for 1 min via the left cardiac ventricle and then perfused 3 min with PFA (4%)-lysine-periodate fixative (PLP). After the kidneys were harvested, they were incubated in PLP solution overnight (38, 39, 42). After five washes in PBS, tissues were cryoprotected in PBS and 30% sucrose overnight before being embedded in Tissue-Tek O.C.T. compound 4583 (Sakura Finetek, Torrance, CA). Cryosections (5 μm) were cut using a Leica CM3050S microtome (Buffalo Grove, IL) and placed onto positively charged slides (Denville Scientific, Holliston, MA). For immunostaining, sections were rehydrated in PBS for 20 min and then permeabilized with 0.1% Triton X-100 for 5 min. After three washes, nonspecific binding was blocked with 1% BSA in PBS for 10 min. The kidneys were incubated with anti-pS256-AQP2 (10 µg/mL) overnight at 4°C. After three washes in PBS, coverslips were incubated with goat anti-AQP2 antibody (C17; 0.4 µg/mL, Santa Cruz Biotechnology) at room temperature. After three washes in PBS, tissue samples were incubated with Alexa 488-conjugated donkey anti-rabbit antibody (2.4 µg/mL) and CY3-conjugated donkey anti-goat antibody (1.9 µg/mL) for 1 h at room temperature. After three washes in PBS, the slide was incubated 8 min with Alexa 647 WGA (0.5 µg/mL). After three rinses in PBS, sections were mounted using Vectashield with DAPI (Vector Laboratories). Images were taken using a Zeiss LSM 800 confocal microscope.