Immunofluorescence (IF) staining.

Mouse kidneys were fixed in 4% paraformaldehyde for 3–4 hours, dehydrated in 30% sucrose, and embedded in Tissue-Tek O.C.T. (SAKURA). Frozen kidney samples were sliced in 5 μm thickness and proceeded to IF staining as previously described (17). In short, kidney sections were permeabilized and blocked with 0.1% Tween 20 in 2% BSA for 1 hour, incubated with rabbit anti-TXNDC5 (1:1500, Proteintech, 19834-1-AP), rat anti-F4/80 (1:1000, Abcam, ab6640), or rabbit anti-CD31 (1:500, Abcam, ab24590) primary antibody overnight at 4°C. After washing, the sections were treated with Alexa Fluor 594–labeled or DyLight 488–labeled anti-rabbit or anti-rat secondary antibodies at room temperature for 1 hour. The sections were then washed and mounted with ProLong Gold (Thermo Fisher Scientific). Measurement of the fluorescence staining area was performed using ImageJ.