Picric acid

For H&E, Sirius red, Masson's trichrome and Periodic acid-Schiff (PAS) staining, tissues were isolated and fixed overnight at 4°C, and treated with 30% sucrose for cryoprotection. Sections of kidney were dewaxed with xylene and a series of descending ethanol gradients. Then sections were stained with Mayer's H&E (Jiancheng) or Sirius Red F3B and a saturated aqueous solution of picric acid (Solarbio, beijing, China), Masson (Jiancheng) or PAS kit (Solarbio), and observed with a Nikon Eclipse Ni-U microscope. For immunohistochemistry (IHC), paraffin sections were stained with anti-Nephrin (Abcam, Cambridge, UK), assessed by light microscopy and quantified using ImageJ (Wayne Rasband, US National Institutes of Health, Bethesda, MD, USA). For β-GAL, TUNEL and immunofluorescence staining, frozen tissue slices were stained with a β-GAL kit (Beyotime, Biotechnology, Shanghai, China), TUNEL kit (Beyotime), or anti-53BP1 (NOVUS, Abingdon, UK), respectively, photographed using an Olympus IX-71 fluorescence microscope and quantified using ImageJ. For DHE staining, 24 h before killing, mice received a 200 μL intravenous injection of dihydroethidium kit (Sigma-Aldrich, St Louis, MI, USA) at 25 mg/kg, and frozen tissue slices were photographed using an Olympus IX-71 fluorescence microscope and quantified using ImageJ.