Galectin 3

Sections of paraffin embedded tissue were subjected to deparaffinization, rehydration, steaming in DAKO target retrieval solution pH 6.1 for 30 minutes, and incubation at room temperature with Protein Block (X0909, DAKO) for 1 h. For immunofluorescent staining, the sections were incubated with primary antibodies to αS (NACP98, 1:500, rabbit polyclonal^{21 (link)}) and Galectin-3 (1:250, mouse monoclonal, Biolegend) at 4 °C overnight, followed by 1.5 hours of incubation with secondary antibodies (1:500) after washing. Non-specific fluorescence signals were blocked by staining with Sudan Black. Sections were coverslipped with Vectashield mounting media (H-1200, Vector Laboratories, Burlingame, CA) and viewed with confocal microscopy.

Kidney cortical tissue was prepared for immunoblot analysis with antibodies against phosphorylated and total Drp1. Kidney cortex samples were homogenized in extraction buffer containing 50 mM HEPES, pH7.4, 150 mM NaCl, 0.5% Triton X-100, 0.025 mM ZnCl2, 0.1 mM Pefabloc SC Plus (Roche, Basel, Switzerland), EDTA-free protease inhibitor cocktail tablet (Roche, Basel, Switzerland), and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Protein concentration was determined using the Micro BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA).
Western blot was performed by separating 15 µg of total protein in 7% SDS-polyacrylamide gels and transferring it onto PVDF membranes (Immobilon-P Millipore, Burlington, MA, USA). Membranes were incubated in skimmed milk blocking solution (5%) for 1 h and incubated overnight at 4 °C with galectin-3 (1:500; 126701, Biolegend, San Diego, CA, USA) in 2.5% skimmed milk. HRP-conjugated anti-mouse IgG antibody was used as secondary antibody. β-Actin antibody (1:20,000; BS1003, Bioworld, Dublin, Ireland) was used as loading control.
Proteins were detected in films (AGFA CURIX, Mortsel, Belgium) after 3-min incubation with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA). Protein bands were quantified by densitometry with the ImageJ software.