Leica dm5500b microscope

Z-stacks spanning approximately 20um were acquired at 20x or 40x with a Leica DFC365 FX digital camera attached to a Leica DM5500 B microscope (Leica, Wetzlar, Germany) using filters for GFP (Ex:450-490, DC:495, EM:500-550) and mCherry (Ex:540-580, DC:595, EM:607-693) and DIC optics for bright field. Images were processed by deconvolution and maximum projection of the relevant Z stacks using LAS X (Leica).

Typically, ∼5 × 10⁴ HUVECs were plated on 0.2% gelatin-coated 4-well chamber slides (Nunc, Thermo Scientific) and grown to full confluence in their growth media at 37 °C. Cells were subjected to immunofluorescence studies as described before (76 (link), 77 (link)). Slides were incubated with conjugated secondary antibodies such as goat anti-rabbit IgG Alexa Fluor® 488 and goat anti-mouse IgG Alexa Fluor® 564 (Invitrogen). Nuclei were visualized with DAPI (Vector Laboratories).
Immunofluorescence images were acquired with a ×63, 1.3 oil immersion objective on a Leica DM5500B microscope equipped with the Leica application suite and advanced fluorescence v1.8 software (Leica Microsystems, Frankfurt, Germany). Confocal analyses were carried out utilizing a ×63, 1.3 oil immersion objective of a Zeiss LSM-780 confocal laser-scanning microscope with Zen imaging software. Images were captured as part of a z-stack series with 3-μm optical slices. A full description of the immunofluorescence and confocal laser microscopy protocol can be found elsewhere (31 (link)).

BOECs were grown on coverslips and then fixed with 4% paraformaldehyde for 15 min at room temperature, and in order to permeabilize the cell membrane, cells were treated with cold methanol for 10 min on ice. Thereafter, 4% normal goat serum was used for blocking for 1 h at room temperature. The cells were then incubated with a mix of anti-Cytokeratin (C2562, 1:250, Sigma-Aldrich) and anti-Vimentin (PLA0199, 1:250, Sigma-Aldrich, USA) primary antibodies in blocking buffer overnight at 4 °C. Negative control was incubated with a blocking buffer lacking any of the primary antibodies. Secondary antibody incubation was done using goat anti-mouse (conjugated to Alexa Fluor 488,1:500) and goat anti-rabbit (conjugated to Alexa Fluor 594, 1:500) secondary antibodies (both Invitrogen, Thermo Fisher Scientific, Eugene, USA) in blocking buffer for 1 h in the dark at room temperature. After incubation, the nuclei were stained using Hoechst 33342 (1:2000, Thermo Fisher Scientific) for 3 min and then the coverslips were mounted with Fluorescence Mounting Medium (Dako, Denmark). Images were taken with a Leica DM5500B microscope equipped with Leica DFC310 camera (Leica, Wetzlar, Germany) and processed with ImageJ (Schindelin et al. 2012 (link)).

BOECs grown on glass coverslips were washed and fixed with 4% paraformaldehyde for 10 min at room temperature, followed by additional fixing and permeabilization with cold methanol for 10 min on ice. After washing, the cells were incubated in blocking solution (4% normal goat serum in PBS) for 1 h at room temperature, and then with anti-Cytokeratin (C2562, 1:250, Sigma-Aldrich) and anti-Vimentin (PLA0199, 1:250, Sigma-Aldrich, USA) antibodies in blocking solution for 1 h at room temperature. Negative control cells were incubated in a blocking solution without primary antibodies. Next, cells were washed three times with PBS and incubated with Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit secondary antibodies (A11029 and A11012, respectively, 1:500, Invitrogen, Thermo Fisher Scientific, Eugene, OR, USA) in blocking solution for 45 min at room temperature in the dark. The nuclei were counterstained with Hoechst 33,342 (Thermo Fisher Scientific), and the coverslips were mounted with Fluorescence Mounting Medium (Dako Glostrup, Denmark). Images were captured with a Leica DM5500 B microscope equipped with Leica DFC310 camera (Leica, Wetzlar, Germany) and processed with ImageJ [52 (link)].