PMC:1189073 / 38984-40368 JSONTXT

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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1189073","sourcedb":"PMC","sourceid":"1189073","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1189073","text":"Immunocytochemistry on MDCK cells.\nMDCK stable cell lines expressing vector alone, wild-type AQP2, or AQP2-F204V (and in some cases transiently expressing a GFP construct) were grown on fibronectin-coated coverslips until tight junctions formed. Cells were treated with or without 150 μM forskolin for 90 min, and fixed in methanol at −20 °C. Subsequently, cells were washed and permeabilized in 0.2% Triton X-100 for 5 min, and sequentially probed for AQP2 and organelle markers for either the PM or the ER. AQP2 was detected using goat anti-AQP2 (1:100; Santa Cruz Biotechnology; #sc-9882) and a 1:200 dilution of AlexaFluor 488-conjugated donkey anti-goat secondary antibody. The PM and ER were probed using mouse anti-Na+/K+-ATPase (Upstate, Waltham, Massachusetts, United States) or rabbit anti-calnexin (Stressgen Biotechnology, Victoria, British Columbia, Canada) antibodies and the secondary antibodies, Cy3-conjugated goat anti-mouse (1:200; Jackson ImmunoResearch, West Grove, Pennsylvania, United States) or AlexaFluor 594-conjugated chicken anti-rabbit (1:200) respectively. Cells were washed in PBS, counterstained with DAPI, and mounted in Vectashield. 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