Lsm 510

As described, the cells grown on chamber slides were transfected with the plasmid, followed by immunofluorescence assay under a confocal laser scanning microscopy (LSM510; Zeiss, Germany). Further, they were cultured with cell-permeant fluorophore MitoTracker Green (Invitrogen/Molecular Probes) or LysoTracker Red (Invitrogen/Molecular Probe) to label mitochondria and lysosomes, and were visualized under a confocal laser scanning microscopy (LSM510; Zeiss, Germany).
The cells cultured on slides were fixed with 4% paraformaldehyde, washed, permeabilized with 0.3% Triton X-100, blocked with 5% BSA, and incubated with primary antibody at 4 °C overnight, and were then incubated with the appropriate secondary antibody. The nuclei were counterstained with 4, 6-diamidino-2-phenylindole (DAPI) (Yeasen, Shanghai, China). Images were taken using a confocal laser scanning microscopy (LSM510; Zeiss, Germany) and the quantification of these images (at least 10 cells/sample) was done using ImageJ software. The mitochondrial network morphology was analyzed using an ImageJ macro tool as described previously^{55 (link)}.

Experiments were carried out using either culture medium or physiological saline solution (PSS) containing [in mM]: 120 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 0.42 Na₂HPO₄, 0.44 NaH₂PO₄, 24 NaHCO₃, and 10 Glucose. Fluorescence signal of Perceval/PercevalHR and pHRed was measured with LSM510 (Carl Zeiss AG, Oberkochen, Germany), which allowed control of CO₂, humidity and temperature while environmental O₂ can also be changed to create hypoxic conditions. Manipulating O₂ tension in the custom made micro-imaging chamber (atmospheric gas) was achieved by an O₂ controller (# 0508.000, PeCon GmbH, Germany). This system has been specifically designed to image cultured cells under conditions of varying O₂ tension, achieved by changing the O₂ level in the atmosphere of the micro-imaging chamber. Images in the experiments of PercevalHR calibration were taken using LSM510 high speed multiphoton confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany). Software analysis of the data from LSM510 and LSM510 Multiphoton were carried out by region of interest analysis using AIM software, version 3.2 SP2 (Carl Zeiss AG).

Freshly isolated VSMCs were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min, washed with PBS, and permeabilized with PBS containing 0.1% Triton X-100 for 20 min at room temperature. Cells were incubated with PBS containing 1% bovine serum albumin for 1 h at room temperature and then were incubated with primary antibodies in PBS containing 1% bovine serum albumin overnight at 4°C. In control experiments, cells were incubated without the primary antibody. The cells were washed and incubated with secondary antibodies conjugated to a fluorescent probe. Unbound secondary antibodies were removed by washing with PBS, and nuclei were labeled with DAPI mounting medium (Sigma-Aldrich). Cells were imaged using a Zeiss LSM 510 laser-scanning confocal microscope. The excitation beam was produced by an argon (488 nm) or helium/neon laser (543 and 633 nm) and delivered to the specimen via a Zeiss Apochromat ×63 oil-immersion objective (numerical aperture, 1.4). Emitted fluorescence was captured using LSM 510 software (release 3.2; Carl Zeiss). Two-dimensional images cut horizontally through approximately the middle of the cells were captured (1024 × 1024 pixels). Raw confocal imaging data were processed and analyzed using Zeiss LSM 510 software. Final images were produced using PowerPoint (Microsoft XP).