2.8. Immunofluorescence (IF) and Confocal Analysis

Cortical neurons (DIVs 14–16) infected with AAV-hSyn-4mtD3mCerulean3+16 or AAV9-hSyn-split-GFP-based contact site sensor (SPLICS_s) were first washed with PBS (once) and then fixed in 4% PFA in PBS supplemented with 0.1 g/mL sucrose. After 15 min in PFA, cells were washed 3 times (3 × 5 min) in PBS and then quenched 20 min with NH₄Cl (50 mM in PBS). Non-infected neurons were stained for cytochrome c for mitochondrial morphology analysis. After the fixation and quenching step, they were permeabilized for 3 min with 0.1% Triton X-100 in PBS and blocked with a PBS solution containing 2% BSA, 10% goat serum and 0.2% gelatin for 30 min. Cells were incubated with primary antibodies diluted in blocking solution (dilution for cytochrome c 1:200) for 1.5 h at RT and washed three times (3 × 5 min) with the blocking solution. Cells were then incubated for 45 min at RT with AlexaFluor 555-conjugated secondary antibodies (Life Technologies; 1:300 dilution in blocking solution). Coverslips were washed 3 times (3 × 5 min) with the blocking solution and then with PBS (10 min); finally, they were mounted with Mowiol.

Images were collected on a Leica TCS SP5 II confocal system using a WLL white laser (Leica), equipped with a PlanApo 100× (numerical aperture 1.4 objective). For all images, the pinhole was set to 1 Airy unit. Confocal microscopy imaging was performed at 1024 × 1024 pixels per image, with a 0.2 Hz acquisition rate. Each channel was collected independently, and photomultiplier gain was adjusted and maintained among different experiments to minimize background and avoid saturation. Once acquired, images were background subtracted and not modified further before analysis. Images were elaborated with ImageJ (National Institutes of Health).

Mitochondrial-ER colocalization: To count ER–mitochondria contacts, a complete z-stack of the cell was acquired every 0.42 µm. Z-stacks were processed using Fiji. Images were first convolved and filtered using the Gaussian Blur filter. A 3D reconstruction of the resulting image was obtained using the Volume J plugin. Two selected faces of the 3D rendering were then thresholded and used to count ER–mitochondria contact sites.

Mitochondrial morphology: Mitochondria labelled with anti-cytochrome c antibody and neuronal staining obtained with NeuroTrace™ 640/660 (Invitrogen, ThermoFisher Scientific) or expressing the 4mtD3mCerulean3+16 were morphologically analyzed as described in [28] using the Fiji software [29]. Briefly, the background was subtracted, and a convolve filter and a mask were applied. Using the software’s analyze particle function, the circularity, the roundness, the aspect ratio (AR) and perimeter of mitochondria were calculated to assess mitochondrial morphology. Results are expressed as the average of values per cell profile.