Observer z1 confocal microscope

Immunofluorescence microscopy was performed by fixing cells with 2% p-formaldehyde (30 min), after which cells were permeabilized with 0.1% saponin in PBS and stained with the anti-GM130 antibody or with AlexaFluor488- or Alexa594-conjugated lectins (1 h at RT). After washing, cells were treated with relevant species-specific Alexa Fluor488- and 594-conjugated anti-mouse or anti-rabbit secondary antibodies. After mounting, cells were imaged using Olympus BX 51 microscope with appropriate filter sets for the dyes. Alternatively, Zeiss Observer Z1 confocal microscope (LSM 700, Carl Zeiss AG, Oberkochen, Germany) with appropriate filter sets, the Zen2009 software, a 63X Plan-Apo oil-immersion objective, was used. Co-localization studies of the mCherry fusion constructs or GT-pHluorin constructs with the Golgi marker (GM130) antibody were done using Alexa488 or Alexa 594-conjugated secondary antibodies, respectively.

Cells were plated on ploy-l-lysine-coated coverslips or in chambered slides (ibidi, 80826) 24 h before treatment or transfection. Where indicated, cells were stained with 100 nM MitoTrackerRed (Molecular Probes MitoTracker Red CMXRos, Thermo Fisher Scientific, M7512) for 1 h at 37 °C and washed twice with PBS before treatment or fixation. After a PBS wash, cells were fixed at RT for 10 min with 3.7% paraformaldehyde. PBS containing 0.05% TritonX-100 was used for permeabilization for 30 min at RT and cells were blocked in 10% FCS in PBS for 30 min at RT. Incubation in primary antibody dilution was carried out at RT for a minimum of 1 h followed by a 1 h incubation with a dilution of fluorophore-conjugated secondary antibodies in the dark. Antibodies were diluted in PBS containing 10% FCS and PBS was used for washing steps. In one of the final washing steps, DAPI was included at a final concentration of 1 μg/mL. Roti-Mount FluorCare (Carl Roth, HP19.1) was used to mount coverslips. Images were acquired with a Zeiss Observer.Z1 confocal microscope (Carl Zeiss) using a 63× oil immersion objective and the ZEN2009 software (Carl Zeiss). Live cell imaging was performed on a Leica sp8 using a 63× glycerol immersion objective and LASX software (Leica). Images were analyzed using Fiji^{93 (link)}.

To study the adhesion of the HeLa cells to Cu-based electrodes, we examined them on a Zeiss Observer.Z1 confocal microscope (Carl Zeiss Microscopy GmbH, Germany) [77 ] using the following protocol. The day before the experiment, the cells were seeded on the electrode surface and incubated under the same conditions as described in the subsection “Biocompatibility study”. Then, the samples were washed three times with PBS and stained with PI diluted by 1000 times. After this procedure, the cells were stained with the Deep Red Cell Mask dye (membrane visualization, Thermofisher Scientific, USA) and fixed with 3.7% paraformaldehyde. Finally, the cells were treated by Fluoroshield with DAPI dye (cell nuclei visualization, Sigma Aldrich, St Louis, MO, USA) and covered with a coverslip that was later glued to the samples by nail polish. In order to obtain high-resolution images of the studied samples, immersion oil was placed between the 100× objective and the coverslip. For analysis, we selected the cells that did not exhibit PI fluorescence and, therefore, were alive before paraformaldehyde treatment.