Anti nephrin

Formalin-fixed and paraffinembedded ZSF1 rat kidney sections were immunostained with anti-nephrin (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) to determine nephrin expression. Donkey anti-rabbit IgG H&L (Alexa Fluor 488) secondary antibody (1:200; Abcam, Cambridge, MA, USA) was used for development with fluorescence quenching liquid (Vector Laboratories). See ESM Methods for details.

Formalin formalin-fixed and paraffin-embedded kidney sections (4μm) were de-paraffinized, re-hydrated, and incubated with rodent declocker solution (Biocare Medical, Concord, CA, USA) at 95°C for antigen retrieval. Kidney sections were then immunostained with anti-nephrin (1:100; Santa Cruz Biotechnology, Inc, Dallas, TX, USA) to determine renal expression of nephrin. Donkey anti-rabbit IgG H&L (Alexa Fluor® 488) secondary antibody (1:200; Abcam, Cambridge, MA, USA) was used for development with fluorescence quenching liquid (Vector Laboratories, Burlingame, CA, USA). Immunostained sections were examined by Nikon 55i fluorescence with a green excitation (200x magnification) and digital images were taken for analysis using Nikon NIS Elements Software. Nephrin expression in the kidney was determined by measuring the percentage of nephrin positive kidney areas. Two observers blinded to the identity of the samples conducted nephrin image analysis in kidney sections. All experiments and data analysis were carried out according to the guideline of the British Journal of Pharmacology (Alexander et al., 2018).

Right naïve kidneys were used to isolate podocytes. Right
nephrectomy was performed at the time of left nephrectomy for XKTx. The porcine
podocyte cultures were developed using a method that we have previously reported
^{3 (link)}. Expression of hCD47
on podocytes was confirmed on the basis of staining with anti-nephrin (Santa
Cruz Biotechnology, Inc.) and anti-human CD47 (Abcam, Cambridge, MA) ab assessed
by immunofluorescence microscopy as well as flow cytometric analysis (FCM. FACS
CANTO II, BD Biosciences, CA) and Reverse Transcription Polymerase chain
reaction (RT-PCR) (see below). In order to assess hCD47 expression by FCM
anti-human CD47 mAbs (B6H12) (BioLegend, San Diego, CA) was used.