Act 2u image analysis software

Paraformaldehyde-fixed kidney was used to prepare paraffin-embedded tissue sample. The paraffin-embedded tissue sections (5-μm thick) were stained with hematoxylin-eosin (H&E) or immunochemistry staining (IHC) (anti-CD68 mAb, Cat. ab31630, Abcam, USA), and observed under a light microscope (Eclipse 80i, Nikon, Tokyo, Japan). For histopathological assessment, representative images were captured with a camera (DS-U1, Nikon, Tokyo, Japan) and analyzed with the ACT-2U image analysis software (version: 1.40.85.221, Nikon, Tokyo, Japan). Tubular injury was evaluated independently by two pathologists blinded to the treatments. Ten fields were randomly selected at a magnification of 400× and 10 tubules were selected from within each field. The scoring was performed as previously described31 (link). Briefly, slides were evaluated based on the presence and extent of tubular epithelial cell flattening (1 point), brush border loss or shedding (1 or 2 points), the presence of a cast (2 points), and caducous necrotic cells without formation of cast or cell debris (1 point). For IHC assessment, ten fields were randomly selected from the slide of each animal at a magnification of 400×, and the number of positively stained cells was counted. Six rats were evaluated for IHC staining in each group.

Formalin-fixed cryosections (10 µM-thickness) were incubated with primary antibodies against Ki-67 (Abcam; 1:100), Keratin 16 (Abcam; 1:250), Keratin 17 (Abcam; 1:100) and CD49f (BD Pharmingen; 1:100) overnight followed by application of the corresponding Alexa-546 or Alexa-555-labeled antibodies (Invitrogen, UK) for 45 min at 37 °C. Cell nuclei were counterstained with DAPI (Vector Labs, UK). Image analysis was performed using a fluorescent microscope in combination with DS-C1 digital camera and ACT-2U image analysis software (Nikon).
For BrdU analysis, the keratinocytes were seeded on collagen-coated sterile glass coverslips in a 6-well cell culture dish and transfected with miR-200c inhibitor or negative control RNA. 48 h after treatment, cells were treated with 10 µM BrdU (Sigma; 2 hours; 37 °C). Next, the cells were fixed with 4% paraformaldehyde (30 min, RT) followed by denaturation in 2 M HCl (30 min, 37 °C) and neutralisation in 0.1 M sodium borate. The cells were stained with FITC-conjugated Anti-BrdU (BD Biosciences; 30 min). Cell nuclei were counterstained with DAPI (Vector Labs, UK). Fluorescent microscopy images from 10 randomly selected fields per coverslip were taken, and the numbers of DAPI+ nuclei and FITC+ nuclei were counted using ImageJ software (NIH, Bethesda, MD USA). Statistical analysis was performed using Wilcoxon rank sum test.