2.1 Unilateral ureteral obstruction model of fibrosis and tissue collection

The animal studies were approved by University of Colorado Institutional Animal Care and Use Committee (IACUC). The University of Colorado animal facility is fully accredited and we followed the National Institutes of Health guidelines for the use of laboratory animals. The experiments use C57/Bl6 6J male mice between 14 and 20 weeks of age. Isoflurane was used as anesthetic, mice were placed prone and chlorhexidine was used to sterilely prep a surgical area. The retroperitoneal area was accessed after a skin incision and after right kidney was exteriorized, the proximal ureter was ligated with a 4-0 silk suture. The wound closed in two layers after the surgical area was cleansed. Kidneys were collected at seven, fourteen, and twenty-one days after the surgery. The left, comparatively uninjured kidneys were used as controls and the right kidneys were used as diseased for each of the time points. The right atrium was cut and mice were perfused with 20 mL phosphate-buffered saline. Kidneys were removed and immersion fixed in 10% formalin for 24 hours. Then kidneys were processed and embedded in paraffin. 5 μm thick sections were cut onto glass slides. For FLIM imaging, slides were imaged by optical sectioning while embedded in paraffin.