Ab300621

The hematoxylin and Eosin Staining Kit (60524ES60, Yeasen, Shanghai, China) was used according to the manufacturer’s instructions. For the presence of morphologic changes, tubular injury was scored as previously reported [10 (link)]. We evaluated the pathology score with two senior pathologists on a blind basis. Each kidney section was analyzed in at least ten fields.
To assess severity of kidney fibrosis, Masson’s trichrome was performed with Masson Stain Kit (G1006, Servicebio, Wuhan, China). It was scored as the percentage of tubulointerstitial fibrosis: no fibrosis (0), less than 25% (1), 25–50% (2), 50–75% (3), and more than 75% (4).
An immunohistochemistry assay was conducted in accordance with standard protocols. F4/80 (1:500, ab300421, Abcam, MA, USA) or α-SMA (1:300, sc-53142, Santa Cruz, CA, USA) were applied to paraffin-embedded sections. Quantification was calculated as a percentage of areas stained positively to total areas.
In the immunofluorescence assay, macrophages were identified with primary antibodies α-SMA (1:500, sc-53142, Santa Cruz, CA, USA) and CD206 (1:200, ab300621, Abcam, Cambridge, UK).

Heart tissue was fixed in 4% POM for >24 h. Part of the heart tissue was paraffin-embedded, while the other part was OCT-embedded. Masson’s trichrome and hematoxylin and eosin (H&E) staining were performed on paraffin sections (4 μm). The OCT sections (8 μm) were treated with wheat germ agglutinin (WGA). Cardiac sections were stained with anti-α-SMA (ab124964, Abcam) by immunohistochemistry at 4°C overnight. The next day, after washing with PBS, the sections were incubated with the secondary antibody and DAB substrate. The color reaction was stopped with ddH2O, and the sections were incubated with hematoxylin. Frozen sections or cells were fixed with 4% POM for 15 min at room temperature and then incubated with anti-CD68 (ab283654, Abcam), anti-CD206 (ab300621, Abcam), or anti-iNOS (ab283655, Abcam) at 4°C overnight. The next day, after washing with PBS, the sections were incubated with fluorescently labeled antibodies at room temperature for 30 min and then with DAPI at room temperature for 3 min. Pictures of the sections were taken at ×100/×200 magnification on a fluorescence microscope (Olympus, BX53, Japan).