Ab40790

For light microscopy, 5-μm sections were cut from paraffin-embedded kidneys and stained with hematoxylin–eosin (H&E) and Masson trichrome. For immunofluorescence, paraffin-embedded kidney sections were deparaffinized in xylene and rehydrated in graded alcohols. Antigen retrieval was performed in boiling Tris-EDTA for 10 min. The slides were washed with Tris-buffered Saline (TBS) then blocked with background buster (NB306; Innovex Richmond, CA) for 1 h at room temperature. Rabbit polyclonal anti-renin antibody (1:200; 54371 AnaSpec, Fremont, CA) or anti-angiotensinogen (1:200; 28101A, Clontech) or anti-prorenin receptor: anti-ATP6IP2 (1:200; ab-40790 Abcam, Cambridge, MA) were added overnight at 4°C and then washed three times with TBS-0.1% Tween 20 (TBS-T), followed by the addition of Alexa 488-conjugated green fluorescent donkey anti-rabbit antibody (1:1000) incubated for 45 min at room temperature. After 5 washes with TBS-T, goat polyclonal anti-AQP2 antibody (1:100; SC-9882, Santa Cruz, Dallas, TX) was applied for 1 h at room temperature followed by Alexa 594-conjugated red fluorescent donkey anti-goat antibody (1:1000) for 1 h in room temperature. Hoechst (1:500) was added to the last wash in TBS at 1:500 and mounted with mounting medium. Slides were examined by confocal laser microscopy and images were all taken at fixed laser settings (Leica, Wetzlar, Germany).

Following antibodies were used for western blot: (P)RR/ATP6ap2 (AF5716, R&D, Minneapolis, MN for mouse tissues and ab40790, Abcam, UK for C2C12 cells), TFEB (Bethyl Laboratories, Montgomery, TX), LC3B, GAPDH, β‐actin, histone 3 (Cell Signaling Technology, Danvers, MA), tubulin, antimouse‐HRP (Santacruz, CA), antirabbit‐HRP and antigoat HRP (R&D, Mineneapolis, MN).

Double immunofluorescence labeling was performed to confirm the presence of PRR in microglial cells [18 (link)]. BV2 and primary microglial cultures grown on glass coverslips were fixed in 4% paraformaldehyde in Dulbecco’s phosphate-buffered saline (DPBS; pH 7.4) and incubated overnight with anti-PRR (1:50; ab40790, Abcam) and anti-Ox42 (1:100; AbD Serotec) primary antibodies. After rinsing with DPBS, the sections were incubated for 150 min with the Alexa-conjugated secondary antibodies (1:200; Molecular Probes; Invitrogen). Finally, cells were stained with Hoechst (10 μg/mL; Sigma), and mounted with Immumount (Thermo Fisher Scientific, Waltham, MA, USA). Immunolabeling was visualized with a confocal laser-scanning microscope (AOBS-SP5X; Leica Microsystems).