Charge coupled device ccd camera

For triple fluorescence analysis, cells were fixed with 4% paraformaldehyde and then permeabilized by 0.5% (v/v) Triton X-100 as reported [32 (link)]. The following primary and secondary antibodies were used: mouse MAb anti-HPV16 E7 antibody (Cervimax), rabbit PAb anti-gelsolin (Novus Biologicals), AlexaFluor 488-conjugated anti-rabbit (Invitrogen), AlexaFluor 488-conjugated anti-mouse IgG (Invitrogen Corporation), and AlexaFluor 594-conjugated anti-mouse IgG (Invitrogen). For F-actin detection, cells were stained with TRITC-phalloidin (Sigma, St Louis MO, USA) for 30 min at room temperature. After washing, all samples were counterstained with Hoechst 33258 (Sigma, 1 mg/ml in PBS) and then mounted in glycerol/PBS (ratio 1:1, pH 7.4). The images were acquired by intensified video microscopy (IVM) with an Olympus fluorescence microscope (Olympus Corporation of the Americas, Center Valley, PA.), equipped with a Zeiss charge-coupled device (CCD) camera (Carl Zeiss, Oberkochen, Germany).

Control and treated cells were fixed with 4% paraformaldehyde (Carlo Erba, Milano, Italia) and then permeabilized by 0.5% Triton X-100 (Sigma-Aldrich). After washings, cells were incubated with the following monoclonal (MAb) or polyclonal (PAb) antibodies, alone or in combination, for 1 hour at 4° C: MAb to mitochondria (Chemicon, Temecula, CA, USA); MAb anti-GD3 (Abcam, Cambridge, UK), PAb anti-MNF2 (Cell Signaling, New England Biolabs, UK), PAb anti-OPA1 (BD Biosciences, Qume Drive, San Jose, CA), and MAb anti-DLP1 (BD Biosciences). After washings, cells were incubated with anti-mouse AlexaFluor 488-conjugated or AlexaFluor 594-conjugated and anti-rabbit AlexaFluor 488-conjugated or AlexaFluor 594-conjugated (all Termo Fischer) for additional 45 min at 37° C. All samples were counterstained with Hoechst 33342 and mounted with glycerol-PBS (2:1). The images were acquired by intensified video microscopy (IVM) with an Olympus fluorescence microscope (Olympus Corporation of the Americas, Center Valley, PA.), equipped with a Zeiss charge-coupled device (CCD) camera (Carl Zeiss, Oberkochen, Germany).