Sirius red

Paraffin-embedded kidney tissue sections (4 μm thick) were stained with Sirius red (all from ScyTek, Logan, Utah, USA) to evaluate the extent of tissue fibrosis. For each kidney section, at least eight fields (at a magnification of × 200) were randomly selected and photographed using a light microscope (BX53F2; Olympus, Tokyo, Japan). The areas of fibrosis and total tissue were measured using ImageJ 1.52d software (Wayne Rasband, National Institutes of Health, USA). A minimum of 10 glomeruli per mouse kidney were evaluated at × 400 magnification. The degree of Sirius red staining of the tubule and glomeruli, as well as the size of the glomeruli, was measured using ImageJ 1.52d software (Wayne Rasband, National Institute of Health, USA)^{37 (link)}.
Paraffin-embedded pancreatic tissue sections (4 μm thick) were stained with periodic acid Schiff (PAS; ScyTek, Logan, Utah, USA) to evaluate morphologic changes. A minimum of 5 pancreatic islets per mouse were selected and evaluated at × 200 magnification using ImageJ 1.52d software (Wayne Rasband, National Institute of Health, USA).

Paraffin-embedded sections were stained with hematoxylin and eosin, Sirius red (ScyTek Laboratories Inc.), or anti-phospho-Histone H2A.X (Cell Signaling, 1:400) with biotinylated IgG and horseradish peroxidase-conjugated ABC reagent (Vector Laboratories, Burlingame, CA). The positive area of Sirius red staining and the number of brown color stained-H2A gamma positive cells were analyzed by Tissue Quest (TissueGnostics). Frozen sections were fixed with cold acetone, and immunostained with anti-AQP3 (Millipore, 1:100), anti-F4/80 (eBioScience, 1:400), or anti-desmin (R&D Systems, 10 μg/ml) antibodies.