Nb 600 749

At the experimental end-point animals were euthanized and tissues were fresh frozen in liquid nitrogen or fixed in 4% paraformaldehyde in PBS overnight. To review histology, slides were stained by haematoxylin and eosin (H&E). Immunohistochemistry (IHC) and immunofluorescence (IF) were performed using standard protocols. Specificity of immunostaining was assessed by incubation in the absence of primary antibody. We used the following primary antibodies: RFP for Strawberry detection (ab34771, 1:400; Abcam), Aquaporin 1 (NB-600–749, 1:500; Novus Biologicals), THP (AF5175, 1:100; R&D), Aquaporin 2 (ab105171, 1:1000; Abcam), Nephrin (AF3159, 1:100; R&D), CD34 (ab8158, 1:50; Abcam), CD73 (AF4488, 1:100; R&D), GFP (ab290, 1:1000; Abcam), HIF2a (NB100-132, 1:150; Novus Biologicals), CA9 (sc-25600, 1:200; Santa Cruz), turbo-RFP (AB234, 1:500; Evrogen) and pS6 (2211, 1:200; Cell Signalling). Secondary antibodies used were conjugated to HRP (IHC) or Alexa-fluor® fluorochromes (IF). Fluorescent images were obtained by confocal laser-scanning microscopy (Leica TCS SP5).

For tissue, frozen sections (5 μm thick) of renal tissue were washed three times/5 min with PBS at room temperature. Slides were blocked using 5% bovine serum albumin for 30 min and then incubated overnight at 4 °C with both STAT3 antibody (CST, USA, 1:200) and the antibody of specific marker, anti-Wilms Tumor 1 (Novus Biologicals, Littleton, NBP2-44607, 1:200) or anti-aquaporin 1 (NB600-749, 1:200), respectively. Slides were then correspondingly incubated with two kinds of secondary antibody (TRITC-labeled antibody from Abcam, ab6786, 1:500; Alexa Fluor488-labeled antibody from Abcam, ab150077, 1:500) at 37 °C for 1 h, and washed by PBS for three times/5 min. The cell nuclei were stained with DAPI for 5 min, and all stained sections were viewed by fluorescent confocal microscopy (Nikon, Tokyo, Japan).