Anti aqp1

Goat serum (10%) was used to block the binding of nonspecific proteins to the sections. Primary antibodies were diluted and incubated with the sections overnight at 4 °C: anti-Aqp1 (Santa Cruz Biotechnology), anti-S100a9 (Proteintech, Rosemont, USA), and anti-Car4 (R&D Systems, Minneapolis, USA). The slides were then washed 3 times with PBS and incubated with secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, USA) for 1 h. The nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich). Images were captured with an IX83 fully automatic inverted fluorescence microscope (Olympus, Tokyo, Japan). Adhesion of neutrophils (S100a9) to Aqp1⁺/Car4^- ECs or Aqp1⁺/Car4⁺ ECs was evaluated by experienced pathologists. For quantification, the numbers of adherent Car4-low ECs and neutrophils per blood vessel were counted. Doublets and triplets were split by considering signal over the average size of a cell. Data were depicted as percentages of Car4-low ECs (number of Car4-low ECs interacting with neutrophils/total number of Car4-low ECs in the blood vessel) and neutrophils (number of neutrophils adhering to Car4-low ECs/total number of neutrophils in the blood vessel) per field. The results represent those from three sections, with nine pictures scanned for each section.

Immunoblot analysis was performed as described previously [16 (link)]. Briefly, each urine sample was loaded with an equal amount of creatinine per lane, and separated by SDS-PAGE. For the detection of each protein, we used the following primary and secondary antibodies: anti-AQP1 (catalog no. sc-20810, Santa Cruz Biotechnology, Santa Cruz, Dallas, TX), anti-AQP2 (catalog no. sc-9882, Santa Cruz Biotechnology), anti-TSG101 (catalog no. ab83–100, Abcam, Cambridge, UK), anti-Alix (catalog no. sc-49268, Santa Cruz Biotechnology), anti-rabbit IgG (catalog no. 7074, Cell Signaling Technology, Danvers, MA), anti-mouse IgG (catalog no. 1858413, Thermo Fisher Scientific, Waltham, MA), and anti-goat IgG (catalog no. P0449, Dako Japan, Tokyo, Japan). Antibody-antigen interactions were visualized using the SuperSignal West-Femto Chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA). Quantitative analysis of the resulting bands was performed using the WinRoof software package version 5.7 (Mitani, Tokyo, Japan).