Sc 7385

The kidney samples were fixed in 10% formalin and embedded in paraffin blocks. Five-micron-thick serial sections were used for immunohistochemistry and laser microdissection. The paraffin sections were stained with periodic acid-Schiff (PAS) or γH2AX (NB100-384, Novus Biologicals, Colorado, USA) and 5-methyl cytosine (sc-56615, Santa Cruz Biotechnology, Texas, USA) antibodies followed by incubation with a peroxidase-conjugated secondary antibody and 3,3-diaminobenzidine tetrahydrochloride (DAB) staining^{9 (link)}. Hematoxylin was used as a nuclear counterstain. Immunofluorescent staining was performed as previously described^{21 (link),30 (link)} using γH2AX, WT1 (sc-7385, Santa Cruz Biotechnology, Texas, USA) and phosphorylated Ataxia Telangiectasia Mutated (pATM) (200-301-400, Rockland, PA, USA) antibodies. Images of all glomeruli without global sclerosis in the biopsy sample were acquired via microscopic examination (400x magnification), and the glomerular area was quantified using color channel analysis and pixel counting using Photoshop CC 2018 software (Adobe) and was divided by the area of the glomerular tuft, as previously described^{21 (link)}.

We isolated mouse glomeruli from mouse kidneys by differential sieving with 180 μm, 100 μm, and 71 μm metal sieves (Retsch) and washing with PBS (Gibco 10010-023). Glomeruli were plated onto a collagen I–precoated flask (Corning 356484) and cultured for 6 days with RPMI media (Gibco 11875-093) before an outgrowth of cobblestone-like cells was observed and harvested. Cells were fixed with 4% paraformaldehyde in PBS (Electron Microscopy Sciences t15710) and washed with ice-cold PBS 3 times before permeabilization with 0.1 Triton X-100 in PBS. Cells were then washed with wash buffer (0.5% BSA, 0.05% Triton X-100 in PBS) twice and blocked with buffer containing 5% goat serum, 1% BSA, and 0.1% Triton X-100 in PBS before incubation with rabbit polyclonal CLVS1 antibody (PA5-32088 Invitrogen) and mouse monoclonal WT1 antibody (sc-7385 Santa Cruz Biotechnology) overnight at 4°C. After additional washes, secondary Alexa Fluor 488 and 568 antibodies were applied (Invitrogen A11008 and A11004, respectively) at a concentration of 1:400 for 1 hour at room temperature. Cells were then washed with wash buffer 4 times before addition of DAPI stain. Immunofluorescence imaging was performed using an EVOS FL imaging system.

Our studied human kidney samples included eight cases of DN, six FSGS, six minimal change disease (MCD) and four normal control tissues. The samples of renal biopsies diagnosed with DN, FSGS or MCD were obtained from the Department of Nephrology, Zhongshan Hospital, Fudan University. The control samples were from healthy portions of nephrectomy specimens which were removed for solitary renal cell carcinoma, and they were further validated by histologic examinations and biochemical performance (urine albumin and creatinine ratio < 30 mg/g). The investigator was blind to the group allocation when immunohistochemistry staining for METTL14 (HPA038002, Sigma), WT1 (sc-7385, Santa Cruz Biotechnology, CA, USA) and Sirt1 (ab110304, Abcam, Cambridge, MA, USA) were administrated on these renal tissues. The investigations were conducted under protocols approved by the Clinical Research Ethics Committee of Zhongshan Hospital, Fudan University after the patients provided their informed consent.