Elyra ps 1 sim microscope

Primary hippocampal neurons were obtained from E18 rat pups. Briefly, 18 000 cells were grown in neurobasal medium supplemented with B27 on poly d‐Lysine coated coverslips. The cells were used for staining at DIV 14–16. The coverslips were fixed with ice‐cold methanol for 10 min, followed by three washes in ddH₂O and PBS. The neurons were then blocked and permeablized with blocking buffer (5% FCS, 0.1% Triton X‐100, and 0.1% glycine in phosphate buffer saline) for 1 h. Next, the neurons were incubated with anti‐mGluR1 or anti‐mGluR5 antibodies, and anti‐Homer1 (cat. no. 160 004, Synaptic systems), diluted in blocking buffer overnight at 4°C. After washing three times in PBS, the cells were incubated with an alexa conjugated secondary antibody for 1 h at room temperature (anti‐rabbit Alexa 488 (1 in 1000), anti‐mouse Alexa 568 (1 in 1000), anti‐Guinea pig Alexa 647 (1 in 1000) (Molecular Probes) and subsequently washed and fixed on glass slides (Superfrost Plus, Thermo) using Moviol. Images were taken using a Zeiss Elyra PS1 SIM microscope with 63× oil immersion lens (N.A. 1.4) and reconstructed images were analyzed using ImageJ.

Visualization of foot process morphology, and analysis of filtration slit density was performed^{57 (link),58 (link)}. In brief, 3 μm paraffin sections mounted on high-precision coverslips coated with Poly-L-lysine (Sigma) were stained for nephrin. Stained sections were mounted with ProLong Gold (Thermo Fisher) for imaging. For visualization super-resolution STED microscopy was performed using a 4 channel STED microscope (Abberior). For quantification, 3D-SIM z-stacks of nephrin-stained kidney sections were acquired with a Zeiss Elyra PS.1 SIM microscope using the ZEN software (all Zeiss, Jena, Germany) with five horizontal shifts and five rotations of the illumination pattern with a slice-to-slice distance of 0.3 µm. Reconstruction of 3D-SIM images was performed using the Zen Black software. PEMP analysis was performed using the PEMP macro for FIJI^{58 (link)}. For this, the capillary area was encircled, and the slit diaphragm length was determined. Filtration slit density (FSD) values were calculated from the ratio of slit diaphragm length and capillary area. Per animal, 12 glomeruli with 31-39 ROIs were analyzed by PEMP.