Kidney immunohistochemistry.
Whole mouse kidneys were fixed in 10% phosphate-buffered formalin for 24 h. Kidneys were embedded in paraffin, and 5-μm sections were prepared. Following antigen retrieval using 10 mM sodium citrate (pH 8) for 10 min at 98 °C, sections were sequentially probed, first for AQP3 and then for AQP2. Sections were incubated in 5% donkey serum and then in goat anti-AQP3 antibody (1:100; Santa Cruz Biotechnology; #sc-9885). Slides were washed with PBS and incubated with AlexaFluor 488-conjugated donkey anti-goat antibody (Molecular Probes, Eugene, Oregon, United States). The slides were subsequently blocked in 5% chicken serum, incubated with a rabbit anti-AQP2 antibody (1:250; USB, Cleveland, Ohio, United States; #A3000–06), which was detected with a AlexaFluor 594-conjugated chicken anti-rabbit antibody (1:500; Molecular Probes). The sections were stained with DAPI and mounted in Vectashield (Vector Labs, Burlingame, California, United States).