Comprehensive Kidney Tissue Analysis

Four μm sections of paraffin embedded kidney tissues were stained with Hematoxylin and Eosin (H&E), Periodic acid–Schiff (PAS), and Trichrome. Tissue damage was examined in a blinded manner and scored as percentage of damaged tubules: 0, no damage; 1, <25%; 2, 25–50%, 3, 50–75%, 4, >75% [15 (link)]. To evaluate renal fibrosis, the fibrotic area and fibrosis intensity in Trichrome-stained kidney sections were quantified with Image J program using published methods by an investigator blinded to the conditions [54 (link)]. To further quantify fibrillary collagen accumulation in the kidney, the kidney sections were stained with Sirius Red/Fast Green Kit (Chondrex, Inc., Redmond, WA) following the kit’s instructions [28 (link)]. Apop Tag red in situ apoptosis detection kit (EMD Millipore, MA, USA) was used for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay following the manufacturer’s protocols. Immunohistochemistry and immunoblotting were performed as previously described [55 (link),52 (link),56 (link)]. The primary antibodies used in this experiment are listed below: rabbit LC3 antibody (Novus Biologicals, CO, USA), mouse monoclonal p62/SQSTM1 antibody (Novus Biologicals, CO, USA), goat neutrophil gelatinase-associated lipocalin (NGAL) antibody (R & D, MN, USA), mouse monoclonal anti-β-actin (Sigma Aldrich, MO, USA).