The human kidney biopsies were processed similarly to the mice biopsies. Histological grading of the AIN was performed in non-scarred cortical areas and graded depending on the extent: grade 0 (affecting < 25%), grade 1 (affecting 25–50%), grade 2 (affecting 50–75%) and grade 3 (affecting > 75%). The presence of ATN was also registered.
For PD-1 immunostaining, 2.5 μm paraffin-embedded kidney sections were dewaxed and rehydrated. Tissue was incubated with mouse anti-human PD-1 antibody (ab52587, Abcam) diluted 1:500. Thereafter, sections were incubated with a conjugated secondary antibody, and binding was revealed using 3,3-diaminobenzidine as a chromogen with avidin-biotin-peroxidase complex (EnVision Flex K8002 visualization System). The presence of PD-1 staining was semi-quantitatively assessed at 400x.
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