Fluorescence-based Analysis of Podocyte Cytoskeletal Changes

Analysis by fluorescence was performed using a Leica confocal microscope DMi8 (Leica Microsystems, Wetzlar, Germany) and images were processed with ImageJ (v. 1.46 r; National Institutes of Health, Bethesda, MD, USA) software. For this, samples were processed as in [59 (link)] and incubated with anti-sirt-1 (1/100). F-actin cytoskeleton distribution was evaluated by phalloidin staining on treated podocytes with HG and Ang II concentrations. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature (RT), washed three times with PBS, permeabilised with 0.1% Triton X-100, washed and incubated with Phalloidin-iFluor 594 Reagent (1/1000, Abcam, Cambridge, UK) for 1 h at RT. Within the last half hour of the incubation period, cells were stained with DAPI. After incubation, cells were washed 3 times in PBS, mounted and observed.