Nephrin

Frozen kidney sections were permeabilized, blocked, and incubated with goat anti-Synaptopodin (Santa Cruz), Nephrin (R&D Systems), rabbit anti-WT1 (Abcam), Ki67 (Abcam), p-Stat3, p-Src, p-Erk, p-H2AX, or NF-κB p65 (Cell signaling), then incubated with Alexa 488- or 594-labeled donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories) or fluorescein labeled Lotus Tetragonolobus Lectin (LTL) (VECTOR) and mounted in DAPI-Fluoromount-G Clear Mounting Media (SouthernBiotech). For antigen retrieval when staining for WT1, p-Stat3, p-Src, and p-H2AX, sections were heated in pH = 6 citrate buffer and permeabilized with Triton x-100 0.2% in phosphate buffered solution (PBS) for 5 minutes, followed by incubation with blocking solution of 5% BSA in PBS at room temperature for 30 minutes. Sections were imaged using an Axio Imager 2 and Observer microscopes (Zeiss). The ratio of cells expressing the protein of interest to total (DAPI + ) cells was calculated for each image. Fluorescence intensity for glomerular staining was measured using Image J and at least 160 glomeruli were evaluated in each group.

Following bioreactor perfusion, kidneys were fixed in 10% neutral buffered formalin (NBF), imbedded in paraffin wax, and sectioned for immunochemistry (IHC). Primary antibodies for IHC were LTL (Vector labs, B1325, Newark, CA, USA), E cadherin (R&D, AF748, Santa Clara, CA, USA), Nephrin (R&D System, AF4269, Toronto, ON, Canada), KSP (Novus Biologicals, NBP1-59248, Littleton, CO, USA), AQP2 (Alomone Labs, AQP-002, Jerusalem, Israel). The primary antibodies were diluted in 0.1%FBS/0.1%Triton/PBS at a 1:100 ratio (except for E cadherin which was diluted at a 1:50 ratio). The secondary antibodies used were: Strepavidin 488 (Invitrogen, S32354, Waltham, MA, USA), Donkey anti-goat IgG Cy5 (Abcam, ab6566, Cambridge, UK), Donkey anti-sheep IgG NL557 conjugated antibody (R&D Systems, NL010), Donkey anti-rabbit IgG594 (Invitrogen, A21207), Donkey anti-rabbit IgG488 (Invitrogen, A21206). They were diluted in the same solution as the primary antibodies at a 1:500 ratio. Cell nuclei were stained with DAPI. Hematoxylin and Eosin (H&E) staining was also done to visualize tissue morphology.
Kidney was also stained with Periodic-acid Schiff (PAS) reagent staining. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay was done as previously described [12 (link)].

For paraffin‐embedded samples, sections were dewaxed and rehydrated, followed by antigen retrieval, blocking with 3% H₂O₂ and serum. For fixed cryosections, staining was started from antigen retrieval and then blocking. Cultured cells were first fixed and blocked with serum. After that, sections or cells were incubated with antibodies. The primary antibodies used were as follows: nephrin (R&D systems, Cat# AF3159, RRID: AB_2155023), podocin (Sigma‐Aldrich, Cat# P0372, RRID: AB_261982), voltage‐dependent anion channel (VDAC; Abcam, Cat# ab14734, RRID: AB_443084), PGC‐1α (Santa Cruz Biotechnology, Cat# sc‐518025, RRID: AB_2755043), and CD36 (Novus, Cat# NB400‐144, RRID: AB_10003498). The Dako EnVision™ Detection Kit (Peroxidase/DAB, Rabbit/Mouse, K5007) was used for immunohistochemistry. Secondary anti‐rabbit, anti‐mouse, and anti‐goat antibodies were used for immunofluorescence. More than 10 fields from each sample were captured for assessment.