Zen 2.5 blue software

Confocal imaging was performed using a ZEISS LSM 800 inverted confocal microscope. To analyse neurite degeneration in MAP2-stained cortical neurons, images were acquired with the 20x objective using tiling function, covering an area of 998.28 x 673.84μM with Z-stacks (20 slices: 19–24μM), including at least 500 neurons in each image. The images were then stitched and a maximum intensity projection of z-stacks were generated. All the confocal imaging and processing were performed using ZEN 2.5 Blue software (ZEISS).
For imaging the axon panels of DRG neurons, immunostained microfluidic chambers were placed carefully on the inverted microscope stage. Images of the axon panels were then taken with a 20x objective using tiling function, covering an area of 607.08 x 603.34μM with Z-stacks (40 slices: 14–18μM). Similarly, for imaging trans-synaptic neuronal cultures in microfluidic chamber, tile images were taken covering an area of 1.76 mm x 606.77μM with Z-stacks (40 slices: 34–40μM).
Live-cell DIC imaging of rabies infected cortical and DRG neurons were performed using Leica SP5 confocal microscope in BSL3. Neurons were cultured in glass bottom dishes (μ-Dish 35 mm high glass bottom, Ibidi), infected with SHBRV lyssavirus at MOI-1 and imaged for 24 hours after infection.

The ± ENZ-treated cells (VCaP: 100 000 per well; 22Rv1: 40 000 per well) were cultured in charcoal stripped medium on 8-well chamber slides (Ibidi GmbH, #80826). Before staining, the cells were treated with EtOH or 100 nM Dex for 1 h at 37°C. For fluorescent staining, the cells were fixed with 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 (PB) for 20 min, and washed with PB. The fixed cells were permeabilized for 15 minutes with 0.1% Triton X-100 and 1% BSA, blocked with 1% BSA for 20 min, and incubated o/n at 4°C with anti-GR antibody (1:500). After washing with PB, the cells were incubated for 2 h with Alexa Fluor 647 labeled secondary antibody (1:500), and nuclei were labeled with 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml) (Sigma-Aldrich, #D8417). The fluorescent images were obtained with a Zeiss Axio Observer inverted microscope (40× NA 1.3 oil objective) equipped with a Zeiss LSM 800 confocal module (Carl Zeiss Microimaging GmbH). Image processing was performed using ZEN 2.5 (blue) software (Carl Zeiss Microimaging GmbH), and the images were quantified with ImageJ software (National Institutes of Health).

Confocal imaging was performed using a ZEISS LSM 800 (ZEISS, Oberkochen, BW, Germany) inverted confocal microscope. Images were taken as Z-stacks and then maximum intensity projection was generated. For imaging neuronal network in microfluidic devices, a tile imaging was performed covering an area of 2.04 mm × 892.84 µm with Z-stacks (39 slices; 39.9 µm) and then stitched together for maximum intensity projection. All the confocal imaging and processing were performed using ZEN 2.5 Blue software (ZEISS, Oberkochen, BW, Germany).