Anti nephrin

Mice were anesthetized using a zoletil-xylazine cocktail during perfusion with phosphate-buffered saline (PBS). Kidney was vertically dissected and fixed with 10% formalin. Kidney sections (3 μm in thickness) were stained with Periodic acid-Schiff (PAS) stain (Sigma). Kidney pathology was evaluated using a lupus nephritis classification system as described in a previous study (16 (link)). Immunohistochemistry was performed using a Vectastain ABC kit (Vector). Tissue sections were incubated with anti-nephrin (Progen), anti-synaptopodin (Abcam), anti-podocin (Abcam), or isotype control antibodies (Abcam) at 4°C overnight. Staining was developed using 3,3′-diaminobenzidine chromogen (Dako). Sections were counterstained with hematoxylin QS (Vector).

Cytosolic proteins were extracted using RIPA buffer containing Halt protease/phosphatase inhibitor cocktail (Thermo). Membrane fraction of cell lysates were extracted using Mem-PER Plus membrane protein extraction kit (Thermo). For immunoblotting, proteins (15–30 μg for each sample) were separated by 10–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes (Biorad) and probed with the following antibodies: anti-pSTAT1_Y701, anti-STAT1, anti-pSTAT3_Y705, anti-pSTAT3_S727, anti-STAT3, anti-p-JAK2, anti-JAK2 (Cell signaling technology), anti-nephrin (Progen), anti-podocin, anti-Na/K ATPase (Abcam) and anti-β-actin (Sigma). Subsequently, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Thermo). Reactive proteins on membranes were visualized using a SuperSignal West Pico Chemiluminescent substrate (Thermo). These membranes were then exposed to an Amersham Imager 600 (GE Healthcare).

The kidney tissue was dewaxed and then heat-mediated antigen repair was performed in sodium citrate solution (pH=6.0) for 15min, and blocked with goat serum. Cultured podocytes growing on a glass slide were fixed in 4% paraformaldehyde for 15 min. The sections and cells were incubated with the following primary antibodies: Prostaglandin D Synthase (PGDS) (ABclonal, Wuhan, China), anti-nephrin (PROGEN, Darmstadt, Germany) at 4°C overnight. Then incubated with the appropriate secondary antibody for 45 or 60 min at room temperature: anti-guinea pig IgG antibody conjugated with Alexa Fluor 568 (Invitrogen, California, USA), anti-rabbit IgG antibody conjugated with Alexa Fluor 633 (Invitrogen). Nuclei were counterstained with Hoechst (Thermo Fisher, Boston, USA). Fluorescence signals were viewed under a fluorescence microscope (Nikon A1R, Tokyo, Japan). NIS-Element (version 5.5) was used to quantify PGDS and nephrin staining intensity.

SDS-PAGE resolved proteins were transferred on PVDF-membranes and detected with target-specific antibodies: anti-Nephrin (Progen #GP-N2, lot 504271, 1:1000), anti-Neph1⁷ (1:250–1:1000), anti-Podocin (Sigma #P0372, lot 035M4851V, 1:1000–2000), anti-ITM2B (Santa-Cruz #sc-50026, lot A2407, 1:200), anti-ATP1A1 (Santa-Cruz #sc-21712, lot L3013, 1:200), anti-HRP-conjugated secondary ABs (Santa-Cruz: sc-2004 (goat anti-rabbit IgG-HRP, lot H1015), 1:25000; sc-2903 (goat anti-guinea pig IgG-HRP, lot I2107, J0812), 1:10,000-1:50,000; sc-2005 (goat anti-mouse IgG-HRP, lot H2014), 1:25000; sc-2768 (rabbit anti-goat IgG-HRP, lot J0713), 1:50,000; Abcam: ab7090 (Goat Anti-Rabbit IgG H&L (HRP) preadsorbed, lot GR270768-25), 1:10,000; or Cell Signalling: 7074 (goat Anti-rabbit IgG, HRP-linked Antibody, lot 28), 1:2000) in combination with ECL Prime (GE Healthcare, Germany) were used for visualization.

Kidneys were frozen in Optimum Cutting Temperature compound (Sakura) and sectioned at 3 μm thickness (Leica Kryostat). The sections were fixed in 4% paraformaldehyde for 15 minutes, permeabilized in 0.5% Triton X-100 in PBS for 5 minutes at room temperature, and then blocked in 2% normal donkey serum for 60 minutes. The sections were then immunostained with the following antibodies: anti-WT1 (catalog BS91456, Bioworld), anti-nephrin (Progen), anti-PP2Acα (catalog ab106262, Abcam), and anti–p-s6 (catalog 4858, Cell Signaling Technology).
The cultured podocytes seeded on coverslips were fixed in cold methanol/acetone (1:1) for 10 minutes at –20°C. After 3 extensive washes with 1× PBS, cells were treated with 1% Triton X-100 for 5 minutes, blocked in 2% normal donkey serum in 1× PBS buffer for 40 minutes at room temperature, and incubated with the following antibodies: anti–p-s6 (catalog 4858, Cell Signaling Technology), anti-CFB (catalog ab192577, Abcam), and anti–p-STAT1 (Ser727) (catalog 8826S, Cell Signaling Technology), followed by staining with FITC- or tetramethylrhodamine-conjugated secondary antibodies: Cy3-AffiniPure Donkey Anti-Rabbit IgG (H+L): 711-165-152, Jackson ImmunoResearch. Cells were also stained with DAPI to visualize the nuclei. Slides were viewed using an OLYMPUS DP74 and BX53 epifluorescence microscope equipped with a digital camera.

Embryoid bodies or kidney organoids were fixed with paraformaldehyde (4%) in PBS for 1 h. Immunofluorescence was performed as previously described (Morizane et al., 2015 (link)). The primary antibodies used were anti-GATA4 (Santa Cruz, #SC-1237), anti-FOXF1 (R&D systems, #AF4798), anti-β III tubulin (Millipore, #MAB1637), anti-OAT1, anti-OAT3, anti-OCT2, anti-KIM1, anti-γH2AX (Cell Signaling, #2577), anti-nephrin (Progen Biotechnik, GP-N2), anti-LTL, anti-podocalyxin (R&D systems, #1658), and Phalloidin (Molecular Probes, #R-415). Alexa 488, 555, or 647 dye-labeled (Molecular Probes; Invitrogen) secondary antibodies were used for immunofluorescence. For 3D whole-mount immunohistochemistry, kidney organoids underwent an additional clearing process after immunostaining as previously described (Hiratsuka et al., 2019 (link)). Immunofluorescence images were obtained using C1 confocal (Nikon) or Stellaris 8 (Leica).