Inversed fluorescent microscope

Morphology of the mitochondrial network was explored using the fluorescent probe MitoTracker Green (0.5 μM, 30 min at 37 °C). After treatment as described above, live cells were washed several times with PBS and maintained in PBS for image acquisition using a Zeiss inversed fluorescent microscope as described above. The quantitative analysis of mitochondrial morphology was performed using a morphometric application programmed on ImageJ^® (National Institutes of Health, MA, USA) [3 (link)]. This application determines the mean area, perimeter, major, and minor radiuses of cell mitochondria. Mitochondrial fusion/fission was evaluated using the form factor (FF = 4π x area/perimeter²) and the aspect ratio (AR = major radius/minor radius) [30 (link)] after top hat filtering was applied to the raw images to remove noise and to obtain a precise definition of the mitochondrial morphology. A minimum of 10 images were analyzed per sample, and three independent series were performed for each treatment.

After treatment as described above, fixed and permeabilized cells (#5 × 10⁴ in glass-bottom 35 mm cells [2 (link)]) were incubated overnight at 4 °C with a binary mixture of anti-IP3R1 (1/500 dilution) and VDAC1 (1/500 dilution) primary antibodies. Thereafter, after two washes with TBS-Tween (tris-buffered saline polysorbate 20) 0.05%, cells were incubated (1 h at 37 °C) with the complementary secondary antibody (rabbit PLUS and mouse MINUS). The proximity ligations were then performed according to the manufacturer’s protocol. Preparations were mounted in Duolink II mounting medium containing DAPI (4′,6-diamidino-2-phenylindole). Fluorescence was analyzed with a Zeiss inversed fluorescent microscope using the AxioVision program. Fluorescence dot signals were quantified using BlobFinder software (v3.2, center for Image Analysis, Uppsala University). Results were expressed as number of blobs per nucleus. A minimum of 10 images were taken per sample, and three independent series were performed for each treatment.