Axiovert 100 lsm510 meta confocal microscope

Cells were grown and co-transfected with pCI-3C/pCI-3Cmut and pCI-EGFP in a black 96-well plate with a transparent bottom (PerkinElmer, Waltham, MA, USA) and analyzed using an Axiovert 100 LSM510 META confocal microscope (Carl Zeiss, Oberkochen, Germany) 18 h p.t. For morphological analysis, the cells were transfected and 18 h later washed with PBS and fixed with methanol (Sigma-Aldrich, Saint-Louis, MO, USA) for 30 min on ice. The cells were washed twice with PBS, incubated for 30 min in blocking buffer (PBS, 5% fetal bovine serum, and 0.25% Triton X-100), and incubated for 20 h at 4 °C in the blocking buffer with goat IgG antibodies specific for the human AIF protein (Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:200. Next, the cells were washed twice with PBS and incubated in the blocking buffer containing donkey anti-goat IgG antibodies conjugated with Alexa Fluor 568 (ThermoFisher Scientific, Waltham, MA, USA) diluted 1:400 for 2 h at room temperature. The cells were washed twice with PBS, stained with 2 µg/mL Hoechst 33342 for 5 min, and analyzed under an Axiovert 100 LSM510 META confocal microscope (Carl Zeiss, Oberkochen, Germany).