Las af software 3

Cells were chemically fixed on slides using a 4% para-formaldehyde solution in PBS. F-actin was stained using Phalloidin-iFluor-647 (ab176759, Abcam, Cambridge, UK) at 1:1000 dilution for 1 h. Nuclear counterstain was performed using 40,6-diamidin-2-phenylindol (DAPI, Vector Laboratories, Burlingame, CA, USA), at 1:1000 dilution. After staining, fixed samples were imaged by a DMI6000CS-TCS SP8 fluorescence microscope (Leica Microsystems, Wetzlar, Germany) with a 20×/0.4 objective using a DFC365FX camera (Leica Microsystems, Wetzlar, Germany) and analyzed with the LAS-AF software 3.1.1 (Leica Microsystems, Wetzlar, Germany).

To detect nephrin and podocalyxin, immunofluorescence (IF) analyses were performed on serial sections of the same biopsies evaluated by IHC. Samples were treated as previously described [20 (link)] and incubated overnight with the appropriate primary antibody diluted in PBS 5% BSA at 4 °C (Table 2). Sections were then incubated with the appropriate fluorescent secondary antibody diluted in PBS 5% BSA at room temperature (Table 2) [20 (link)]. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA) diluted 1:1000 in PBS. Negative controls were run by omitting primary antibody. Images were acquired with a DMI6000CS-TCS SP8 fluorescence microscope (Leica Microsystems, Wetzlar, Germany) with a 20×/0.4 objective using a DFC365FX camera (Leica Microsystems, Wetzlar, Germany) and analyzed with the LAS-AF software 3.1.1 (Leica Microsystems, Wetzlar, Germany).