4.7. Immunostaining of Transduceosome/Metabolon Protein

C20, HMC3 and U87MG cells were cultured on a round glass coverslips with a density of 3 × 10⁴ cells/well per 24-well plate. Cells were stained with addition of 500 nM MitoTracker^TM Red CM-H₂Xros (ThermoFisher) diluted in cell medium for 45 min at 37 °C, before fixation with 4% of PFA for 15 min followed by three washes with PBS. Then the cells were permeabilized with a PBS solution containing 2.5% BSA and 0.3% Triton X100 for 10 min, blocked with the same solution without Triton for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibody diluted in blocking solution (anti-TSPO 1:1000; anti-CYP11A1 1:100; anti-StAR 1:100). The day after, cells were washed with PBS, and incubated for 2 h with an anti-rabbit goat IgG conjugated with an Alexa Fluor 488 dye (Thermo Fisher Scientific, 1:100). After washes, cell nuclei were stained with DAPI (Sigma Aldrich) 1:5000 diluted in blocking solution. Coverslips were mounted with Vectashield (Vector Labs) on a microscope support and imaged with an epifluorescence microscope (Nikon E-Ri) using a 60X oil objective (NA 1.4). Sequential z-stacks in the blue, green, and red emission channels were acquired, respectively, for the detection of DAPI, TSPO/StAR/CYP11A11, and mitochondria at 60× magnification. Exposure time and acquisition parameters were kept constant for analysis of the green channel in the three different cell lines. The generated stacks were analyzed by using ImageJ program ImageJ (nih.gov). The z-projections of the stacks were background-subtracted, subjected to linear brightness/contrast enhancement, and merged for co-localization analyses.