Apotome 2 microscope

HUVEC were grown on gelatin (#TC041, HiMedia)-coated coverslips up to 70% confluency. After completion of treatment, they were washed with PBS and subjected to fixation with 4% ice-cold paraformaldehyde. Cells were incubated with 0.1% Triton X for permeabilization. Cells were then incubated with BSA (1%) for 1 h, followed by overnight incubation with EZH2 antibody (1:1000, #5246, Cell Signaling Technology). Cells were washed with PBS to remove unbound primary antibody, and were subsequently incubated with Alexa fluor 555 secondary antibody (1:4000; #A32732, Thermo Fisher Scientific) for 2 h. To stain F-actin, cells were incubated with rhodamine-tagged phalloidin (1:5000; #R415, Thermo Fisher Scientific) for 30 min, followed by incubation with DAPI (#D9542, Sigma-Aldrich) for nuclear staining. Fluorescence images were captured using a Zeiss ApoTome.2 microscope (Carl Zeiss, Jena, Germany), and intensities were measured using ImageJ software.

For Cx43 immunodetection, 10 µm thick cryosections of myocardial apex tissue were used. According to our previous publications [59 (link),61 (link)], cryosections were fixed in ice-cold methanol, permeabilized in 0.3% Triton X-100, blocked in solution of 1% bovine serum albumin and incubated with primary anti-Cx43 antibody (diluted 1:500, CHEMICON International, Inc., Temecula, CA, USA, #MAB 3068 and secondary antibody with FITC-fluorescein isothiocyanate (diluted 1:500, Jackson Immuno Research Labs, West Grove, PA, USA, #111-095-003). For actin filaments, visualization cryosections were stained with phalloidin (Sigma-Aldrich, St. Louis, MO, USA, #P 2141). Microscopic images were captured by Zeiss Apotome 2 microscope (Carl Zeiss, Jena, Germany). Quantification of Cx43 immunofluorescence signal was performed on a 15 randomly selected myocardial area (per heart) and expressed as total integral optical density per area (IOD) [62 (link)].

Gentamicin sulfate salt (Sigma-Aldrich) was mixed with the Sulfo-Cyanine3 NHS ester (Lumiprobe) in 50:1 molar ratio and incubated for 1 h at room temperature. The conjugate (Gen-Cy3) was isolated by reversed-phase chromatography (column C18), aliquoted, dried, and stored in the dark at -20°C. Prior to usage, the conjugate was resuspended in sterile water, absorbance at 548 nm was measured, and a concentration was calculated using the molar attenuation coefficient of the Sulfo-Cyanine3 NHS ester. In the cell labeling experiments, gentamicin sulfate used in a protection assay was replaced with Cy3-conjugated gentamicin (GEN-Cy3) at the indicated concentrations. CHO WT and CHO ΔXylT cells incubated with GEN-Cy3 were fixed at different time points post infection.
CHO WT and CHO ΔXylT cells were seeded on coverslips and then infected with S. Typhimurium eGFP at an MOI of 50. Upon bacterial invasion, CHO cells were incubated for 2, 7, or 24 h in presence of 50 nM Lysotracker Red DND-99 (Sigma-Aldrich). Then, CHO cells were extensively washed with PBS, fixed with 4% paraformaldehyde (PFA) and stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) (1:1000) to visualize nuclei. Images were recorded on a Zeiss Apotome.2 microscope using AxioVision 4.9.1 software (Zeiss).