R37120

Recombinant human FABP4 was labeled with Alexa Fluor 647 reactive dye (AF647, A37573, Thermo Fisher Scientific, MA) according to the manufacturer’s protocol^{26 (link)}. Ten minutes after intravenous injection of AF647-FABP4 (15 μg/mouse in 200 μl of saline) or saline alone, kidneys were isolated, longitudinally cut into two equal halves, fixed in Carnoy’s solution (60% ethanol, 30% chloroform and 10% glacial acetic acid) for 6 hours and embedded in paraffin. Immunofluorescence analysis was performed with fluorescein LTL (FL-1321, Vector laboratories, CA) or primary antibodies against THP (sc-19554, Santa Cruz, TX) or CalD (ab82812, abcam, MA). Anti-goat and anti-mouse secondary antibodies conjugated with Alexa Fluor 488 (A-110055 and R37120, respectively, Thermo Fisher Scientific, MA) were used to detect THP and CalD, respectively. Images for immunofluorescent analysis were captured with Biozero-8100 immunofluorescence microscope (Keyence Corporation, Osaka, Japan).

For immunofluorescence staining, HUVECs were fixed using 4% paraformaldehyde (PFA) in HBSS+ for 10 min at room temperature. The fixative was aspirated, and the cells were rinsed once with HBSS+. Next, the cells were permeabilized for 2 min with 0.2% Triton X-100 in HBSS+ and washed once with HBSS+. The cells were blocked in 5% BSA in HBSS+ for 30 min and incubated with the primary antibody solution overnight at 4°C. Mouse anti-human CD144 (1:150; 555661, BD Biosciences, USA), sheep anti-human CD31 (1:150; AF806, R and D Systems, The Netherlands) and rabbit anti-human vWF (1:1000; A0082, Agilent Dako, USA) were used as the primary antibodies. The cells were washed with HBSS+, followed by an one-hour incubation with Hoechst (1:2000; H3569, Invitrogen, USA), rhodamine phalloidin (1:200; P1951, Sigma-Aldrich, The Netherlands) and the secondary antibody solution, containing Alexa Fluor 488-conjugated goat anti-mouse (1:250; R37120, ThermoFisher, USA), Alexa Fluor 488-conjugated donkey anti-sheep (1:250; A11015, ThermoFisher, USA) and Alexa Fluor 647-conjugated goat anti-rabbit (1:250; A27040, ThermoFisher, USA) antibodies. The cells were washed three times with HBSS+. High-quality Z-stack images of the stained cells were acquired using a high-content confocal microscope (Molecular Devices, ImageXpress Micro Confocal).