Zen black software

GBM tumorosheres in U-bottom well plate were imaged from 4 h to 48 h after addition of NK cells under ×100 magnification using inverted laser scanning confocal microscope with AiryScan LSM880 and ZEN black software (both Zeiss, Germany). CellTracker blue was excited with laser line at 405 nm and emission was detected between 410 and 485 nm. CellTracker green and PI were excited with laser line at 488 nm and emission was detected between 495 and 550 nm for CellTracker green and 570–700 nm for PI. Laser power, gain and offset were kept constant between experiments and conditions. Z-stacks of confocal sections were acquired with a step size of 2.99 μm. Z-stack images of tumorospheres were analyzed and reconstructed using ZEN blue software (Zeiss, Germany) and Imaris (Bitplane) software version 9.5.1 (Oxford Instruments, United Kingdom). The number of GBM cells and dead cells was quantitated with the spot detection tool of the Imaris software. For analysis of direct cellular interactions, surface 3D renderings were created using surface area module for NK cell (CellTracker blue) and GSLC (CellTracker Green) surface using Imaris software. We then obtained surface reconstructions of NK cell surface touching GSLC surface in gray color using surface-surface contact area plug in.

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.

Labeled cells were imaged using an LSM 780 confocal microscope (Carl Zeiss). Images were processed using Photoshop CS2 software (Adobe Systems). Choroidal vascular density was analyzed using ImageJ software (NIH) as described previously (Le et al., 2010 (link)). Fluorescence intensity was quantified using Zen Black software (version 2012; Carl Zeiss) according to the manufacturer’s instructions. All images shown are representative of three to eight independent experiments.