Micrographs of PAS-stained sections were taken with an Olympus BX50 microscope equipped with an Olympus UC30 camera. 10x (NA 0.25) and 40x (NA 0.6) objectives were used.
For confocal laser scanning microscopy a Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany) equipped with a 63x (NA 1.4) oil immersion objective was used. Single micrographs of each glomerulus were acquired with 0.240 µm/pixel and subsequently, plan view areas of the glomerular capillary surface, which were positive for nephrin, were imaged with 0.080 µm/pixel.
For SIM a Zeiss Elyra SP.1 system (Zeiss Microscopy, Jena, Germany) equipped with a 63x (NA 1.4) oil immersion objective was used. Z-Stacks with a size of 2,430 × 2,430 pixels2 (link) (78.35 × 78.35 µm2) with a slice-to-slice distance of 0.3 µm were acquired over approximately 4 µm using the 561 nm laser, with 2.4% laser power and an exposure time of 100 ms. The 34 µm period grating was shifted 5 times and rotated 3 times on every frame. The 3D SIM reconstruction was performed with the Zeiss ZEN Software using following parameters: Baseline Cut, SR Frequency Weighting: 1.3; Noise Filter: −5.6; Sectioning: 96, 84, 83.
Parts of the renal biopsies were fixed in 2.5% glutaraldehyde and embedded in Glycidether 100 (formerly called Epon 812). Ultrathin sections of 70–90 nm were cut with a Leica ultratome equipped with a diamond knife, stained with uranyl acetate and lead citrate. The pictures were examined with a Libra 120 electron microscope from Carl Zeiss (Zeiss Microscopy, Jena, Germany). For deconvolution analysis of the wide field image stacks, ZEN 2.3 blue edition (Zeiss Microscopy, Jena, Germany) image processing software was used.
For confocal laser scanning microscopy a Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany) equipped with a 63x (NA 1.4) oil immersion objective was used. Single micrographs of each glomerulus were acquired with 0.240 µm/pixel and subsequently, plan view areas of the glomerular capillary surface, which were positive for nephrin, were imaged with 0.080 µm/pixel.
For SIM a Zeiss Elyra SP.1 system (Zeiss Microscopy, Jena, Germany) equipped with a 63x (NA 1.4) oil immersion objective was used. Z-Stacks with a size of 2,430 × 2,430 pixels2 (link) (78.35 × 78.35 µm2) with a slice-to-slice distance of 0.3 µm were acquired over approximately 4 µm using the 561 nm laser, with 2.4% laser power and an exposure time of 100 ms. The 34 µm period grating was shifted 5 times and rotated 3 times on every frame. The 3D SIM reconstruction was performed with the Zeiss ZEN Software using following parameters: Baseline Cut, SR Frequency Weighting: 1.3; Noise Filter: −5.6; Sectioning: 96, 84, 83.
Parts of the renal biopsies were fixed in 2.5% glutaraldehyde and embedded in Glycidether 100 (formerly called Epon 812). Ultrathin sections of 70–90 nm were cut with a Leica ultratome equipped with a diamond knife, stained with uranyl acetate and lead citrate. The pictures were examined with a Libra 120 electron microscope from Carl Zeiss (Zeiss Microscopy, Jena, Germany). For deconvolution analysis of the wide field image stacks, ZEN 2.3 blue edition (Zeiss Microscopy, Jena, Germany) image processing software was used.
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