Tcs sp5 ds

SKMel37.B7H6 cells were incubated with IncuCyte® CytoLight Rapid Green or Red Reagent (Essen Bioscience), washed and plated in microscopy Nunc™ Lab-Tek™ Chamber Slides (Thermo Fisher Scientific) to adhere. Afterward, medium was removed, and effector cells were added in RPMI medium without phenol red (Thermo Fisher Scientific) and the co-cultures were incubated for 24 h at 37°C. Images were acquired with a LEICA TCS SP5 DS (Leica) confocal microscope and analyzed with the Fiji software (http://fiji.sc/Fiji).

Confocal images for phenotype evaluation were acquired using a confocal fluorescence microscope (DM6000 B) with a scanner (Leica TCS SP5 DS) utilizing a 20 × 0.7 objective, 1024× 1024 pixels and 1.5 μm Z-steps. For evaluation Leica Application Suite X, Gimp 2 and ImageJ were used.
Alterations in larvae retinal hyaloid vasculature was imaged at 5 dpf, at which point larvae were anaesthetized in 0.003% tricaine and fixed in 4% PFA/PBS for 24 h at 4 °C. Fixed larvae were washed three times for 15 min in PBS at RT before incubation in 0.25% Trypsin/EDTA solution (Gibco) buffered at pH 7.8 with TRIS (1.5 m, Roth) for 80 min at 37 °C. Afterwards larvae were washed three times for 15 min and stored in PBS until preparation. The larvae retinal hyaloid vasculature was dissected under a stereoscope and visualized with the confocal fluorescence microscope as described above [73 (link)].
For imaging of the zebrafish adult retinal vasculature, retina dissection and analysis were performed as recently described [74 ]. Fixated eyes were transferred to an agarose platform covered with PBS. The retina was detached from the eye and washed before it was transferred on a slide, immersed in mounting media and covered with a cover slide. Pictures were taken using the confocal fluorescence microscope as described above.

For imaging of the zebrafish neurogenesis, Tg(hb9: GFP) larvae were anesthetized in 0.0003% tricaine and dechorinated at 24 hpf, 48 hpf, respectively and fixed in 1% low melting gel for scanning. Confocal images for phenotype evaluation were acquired using a confocal microscope (DM6000 B) with a scanner (Leica TCS SP5 DS) utilizing a 20 × 0.7 objective, 1,024 × 1,024 pixels, 0.5 μm Z-steps. The motor axon length at a defined location in the intersomitic segments was measured for 4–6 axons per larva by blinded observer using ImageJ software with neural tracking plugin (Vaccaro et al., 2012 (link); Ciura et al., 2013 (link); Pagnamenta et al., 2021 (link)). The axon length was measured in detail (Supplementary Figure 1).