Elyra ps1 system

Structured illumination microscopy (SIM) was performed on a Zeiss ELYRA PS.1 system (Carl Zeiss Microscopy GmbH, Goettingen, Germany) equipped with an Andor EM-CCD iXON DU-885 with 1004 × 1002 pixels. Z-stacks were taken using a 63x oil immersion objective with a numerical aperture of 1.46. To generate structured illumination a grid pattern was projected onto the image plane in five different positions and at five different modulation angles to obtain high frequency information within the low frequency information captured by the optical system. For the Cy3-channel back-computation of the lower frequencies using Fourier transformation was performed using the Zeiss ZEN Structured Illumination Processing tool to increase the resolution in the final image. Due to the almost homogeneous distribution of the eGFP signal there was no gain in resolution by image processing in the eGFP channel.

For confocal laser scanning microscopy, a Leica TCS SP5 (Leica Microsystems) equipped with a 63 × (NA 1.4) oil immersion objective was used. Mouse glomeruli were imaged with 6.080 pixel/µm. Human glomeruli were imaged with 5.853 pixel/µm. For 3D‐SIM a Zeiss Elyra PS.1 System (Zeiss Microscopy) equipped with a 63 × (NA 1.4) oil immersion objective was used. Z‐Stacks with a size of 1280 µm × 1280 µm with a slice‐to‐slice distance of 0.2 µm were acquired over approximately 4 µm using the 561 nm laser, (3% laser power, exposure time:100 ms) and the 488 nm laser (4% laser power, exposure time: 100 ms). The 28 µm period grating was shifted five times and rotated five times on every frame while acquiring widefield images. The 3D‐SIM reconstruction was performed with the Zeiss ZEN Black Software using following parameters: Baseline Cut, SR Frequency Weighting: 1.0; Noise Filter: −5.6; Sectioning: 96, 81, 81. ZEN Blue software was used for maximum intensity projections.

To obtain confocal laser scanning micrographs, a Leica TCS‐SP5 system was used. Micrographs were acquired using a 40× 1.2 NA oil immersion objective with a voxel size of 189 × 189 × 500 nm (xyz). For whole‐slide near‐infrared imaging, an Olympus FV3000 system with a 20×, 0.8 NA air objective equipped with a 405, 488, 561 and 640 nm laser lines and an external NIR‐unit with a 730 nm laser line was used. The whole section was imaged as tiled stacks over 2 µm with a voxel size of 222 × 222 × 500 nm. Tile data were stitched within the Olympus FV3000 CellSense software. For super‐resolution 3D‐structured illumination microscopy, a Zeiss Elyra PS.1 system (Carl Zeiss Microsystems) or a Nikon N‐SIM‐E was used as described before.³ Whole‐slide images of PAS‐stained sections were acquired on a Leica SCN400 slidescanner. SCN files were imported and processed with QuPath (v0.3.0).

Sample processing and subsequent imaging was performed as described before (Artelt et al., 2018 (link)). In short, 4 µm paraffin sections were directly mounted on coverslips (VWR). To correct for paraformaldehyde-induced autofluorescence, samples were incubated with 100 mM glycine in PBS for 10 min. Samples were blocked with 1% (v/v) fetal bovine serum, 1% (v/v) goat serum, 1% (v/v) bovine albumin and 0.1% (v/v) cold fish gelatin in PBS at room temperature for 1 h. Primary antibodies against nephrin (guinea pig, Progen GmbH, 1:100) and SSeCKS (Table S1; 1:200) were diluted in blocking solution and detected by a secondary anti-guinea pig antibody (1:800) and anti Cy3-labeled polyclonal goat anti-rabbit IgG (H+L) (1:600) (both from Jackson ImmunoResearch, Hamburg, Germany) diluted in blocking solution. Three-dimensional structured illumination microscopy (SIM) images were acquired using a Zeiss Elyra PS.1 system. Using Zeiss ZEN black software, 3D SIM images were reconstructed. Podocyte PEMP was performed using FIJI and a custom-build macro (Siegerist et al., 2017 (link)). Analysis was performed in two different MWF rat glomeruli. FSD was measured in eight (four sclerotic and four non-sclerotic) glomeruli.

Imaging was performed using a Zeiss Elyra PS1 system. 3D-SIM data was acquired using a 63×1.4NA oil objective. 488, 561, 642 100 mW diode lasers were used to excite the fluorophores together with respectively a BP 495-575+LP 750, BP 570-650+LP 75 or LP 655 excitation filter. For 3D-SIM imaging a grating was present in the light path. The grating was modulated in 5 phases and 5 rotations, and multiple z-slices were recorded with an interval of 110 nm on an Andor iXon DU 885, 1002×1004 EMCCD camera. Raw images were reconstructed using the Zeiss Zen software. Examples are shown in Fig. S5.