Picrosirius red

Kidneys were fixed in 10% neutral buffered formalin and embedded in paraffin. 5 ~m sections were cut for hematoxylin and eosin (H&E) and Masson’s trichrome (MTS) stainings (Leica). picrosirius red staining for collagen was performed using 0.1% picrosirius red (Direct Red 80, Sigma) and counterstained with Weigert’s hematoxylin. The extent of renal injury was estimated by morphometric assessment of tubular damage and interstitial fibrosis. To estimate the protection from tubular damage in UUO, NTN and folic acid mice, eight 200× visual fields were randomly selected for each slide and the number of healthy tubules was manually counted using the count tool of Adobe Photoshop. Tubules were defined as healthy when the dimension, structure, relative nucleus–cytoplasm disposition, integrity of the brush border and of the basal membrane resemble those of normal tubules from healthy kidneys. For the analysis of the interstitial fibrosis in UUO, NTN and folic acid mice, eight 200× visual fields were also randomly selected for each MTS and picrosirius red stained kidney section and interstitial fibrosis was manually evaluated by a grid intersection analysis using Adobe Photoshop. Representative images were acquired with Leica DM 1000 LED microscope and the MC120 HD Microscope Camera with Las V4.4 Software (Leica).