C2 confocal system

Microinjected embryos were anesthetized with 0.04% tricaine (Sigma-Aldrich, E10521), embedded in 0.8% low-melting agarose prepared in fish water and lateral mounted on a depression slide. A Nikon C2 confocal system was used to record images that were analyzed with Fiji (ImageJ) software. Dynamic of interaction between AMBRA1 and mitochondrial outer membrane was studied by time-lapse confocal microscopy analysis of 32-hpf (hours post fertilization) zebrafish embryos treated with 488 nm wavelength laser (at 1/10 of the maximum intensity) for 2 s every 30 s over a period of 3 min. Acquisitions were made every 30 s in both green and red channels.
For functional experiments, microinjected embryos images were captured every 2 min over a period of 8 h from 34 hpf to 42 hpf with the same laser’s settings.

After the exposure to samples and controls, ENZO Lyso-ID red detection kit (1:1000; Enzo Life Sciences, Inc., Lausen, Switzerland) together with 1 µg/mL Hoechst 33,342 were added for 20 min at RT. After removal of the staining solution, cells were rinsed once with Kit buffer. After switching to PBS, cells were immediately imaged with a HCS system based on motorized Nikon Ti2 E inverted microscope with C2 confocal system (Nikon CEE GmbH) using Andor Zyla VSC-08691 camera. Hoechst 33,342 was detected at ex 395 nm/em 432 nm and LysoID at ex 555 nm/em 596 nm. Z-stacks were taken at 0.5 µm and 2500 cells were analyzed for number of lysosomes/cell using NIS Elements software with JOBS module. Histograms and significances generated by analysis of 10,000–100,000 DMBM-2 macrophages were compared and no differences regarding significant changes identified. Analysis of 15,000 (THP-1), 30,000 (RAW264.7, J774, MDM), and 40,000 (DMBM-2) cells is presented. For identification of the correlation between intensity and area in the LysoID staining, Spearman’s rank correlation is given. Coefficients between 0.5 and 0.7 were regarded as moderate correlation [48 (link)].