Lsm 510 meta argon krypton laser scanning confocal microscope

Freshly dispersed ECs (as above), together with residual VSMCs were left for 1hr before use. Cells were then fixed in 4% paraformaldehyde (Sigma-Aldrich, United Kingdom) in PBS for 20 min at RT as previously described (Barrese et al., 2018a (link)). Cells were treated with 0.1 mol-L^–1 glycine for 5 min and incubated for 1 h with blocking solution (PBS-0.1% Triton X-100-10% bovine serum albumin) at RT. Following incubation overnight at 4°C with primary antibodies (Table 2) diluted in blocking solution (anti-PECAM-1 for ECs, anti-α-actin for VSMCs and anti-K_V7.1, K_V7.4, and K_V7.5 channel for ECs/VSMCs), cells were then washed for 20 min with PBS, incubated for 1 h at RT with the secondary conjugated antibodies diluted in blocking solution. Excess secondary antibody was removed by washing with PBS and cells mounted using media containing 4′,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining. Using triple staining, ECs and VSMC were differentiated via the following: ECs were positive for anti PECAM-1 and negative for anti-α-actin; while VSMC was positive for anti-α-actin and negative for anti-PECAM-1 (data not shown). Cells were analyzed using a Zeiss LSM 510 Meta argon/krypton laser scanning confocal microscope (Image Resource Facility, St George’s University, London).

For immunofluorescence analysis, HeLa cells were plated on glass coverslips. Cells were fixed with 3% paraformaldehyde (PFA), permeabilized with 0,1% v/v Triton, and immunostained with the indicated antibodies. Immunofluorescence was visualized using a Zeiss LSM 510 Meta argon/krypton laser scanning confocal microscope (Oberkochen, Germany). Quantification of the immunofluorescent images and correlation (Pearson’s) coefficient were calculated by the ImageJ software (NIH, Bethesda, MD, USA).

For immunofluorescence study, HEK293 cells were plated on poly-l-lysine coated (10 μg/ml) glass coverslips. Cells were fixed and immunostained with ant-KSR1 (1 : 100) and anti-praja2 (1 : 500) antibodies for the endogenous protein levels, with anti-Flag (1 : 500) antibody for the peptides. Immunofluorescence was visualized using a Zeiss LSM 510 Meta argon/krypton laser scanning confocal microscope (Oberkochen, Germany). Quantification of the immunofluorescent images and correlation (Pearson's) coefficient were calculated by the Image-J software (NIH, Bethesda, MD, USA).