Confocal Microscopy of Podocyte Cytoskeleton

Confocal microscopy was performed on RPCs cultured on Softslip by using a Leica SP5 AOBS confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Chameleon Ultra-II two-photon laser (Coherent, Milan, Italy). Cells were fixed for 10 min with paraformaldehyde. The following antibodies were used: Anti-paxillin (ab32084, dilution 1:250, Abcam, Cambridge, UK), anti-nephrin (NMP1-30130Y17-R, dilution 1:200, Novus Biologicals Centennial, CO, USA), anti-ROCK1 (Cat# 202694T02, dilution 1:200, Sino Biological, Wayne, PA, USA), anti-ROCK2 (Cat# HPA044109, dilution 1:30, Sigma Aldrich, Milan, Italy). Staining with Alexa Fluor 546 phalloidin (Cat# A22283, dilution 1:100, Life Technologies, Monza, Italy) was performed following manufacturer’s instructions. Double immunolabelling was performed as described [13 (link)] and Alexa-Fluor secondary antibodies were from Molecular Probes (Life Technologies). Nuclei were counterstained with DAPI (Life Technologies) and excited with multiphoton laser at 800 nm.