Anti nfat5

Corneas were removed and immediately fixed with acetone, followed by blocking in 2% BSA. Corneas were stained with Alexa Fluor-647 anti-vimentin (Abcam, Cambridge, UK), anti-F4/80 (Invitrogen, Eugene, USA), anti-NFAT5 (Thermo Fisher, USA) or anti-LYVE1 (Abcam, UK). Secondary antibodies for staining included Alexa Fluor 555 goat anti-rat and Alexa Fluor 488 goat anti-rabbit (Invitrogen, USA).

The expression of Flotillin-1, NGAL, NFAT5, and HIF-1alpha were studied by Western blot according to standard conditions (45 (link)). Briefly, the proteins were separated on 7.5–10% polyacrylamide gels and transferred to nitrocellulose membranes (Invitrogen Carlsbad). Then, the membranes were blocked using 5% milk for 45 min at room temperature. Then, the membranes were probed with anti-Flotillin-1 (Abcam, Ab133497), anti-Lipocalin-2/NGAL (R & D Systems, Inc, MAB-1757), anti-NFAT5 (Thermo Scientific, PAI-023), or anti-HIF-1α (Abcam, Ab113642) antibodies overnight at 4°C. Following three washes with 0.1% Tween-20 in phosphate-buffered saline, the membranes were incubated with secondary [anti-mouse IgG-Alexa Fluor 750 (Thermo, A21037) or anti-rabbit IgG (Alexa Fluor 750, Thermo, A21039)] antibody in a 1:15,000 dilution for 2 h at room temperature. The infra-red fluorescence (IR) imaging was quantified using the Odyssey-CLx (Li-Cor) equipment and the software Image Studio Lite version 5.25 (Li-Cor).