Alexa fluor 594 labelled goat anti rabbit igg

Phase-contrast and fluorescence microscopy was performed as described in Pazos et al.[26] (link) using an Olympus U-MNIBA2 filter (excitation filter: 470–490 nm; emission filter: 515–550 nm; dichroic filter: 505 nm) to visualize the GFP and YFP fluorescence [29] (link). Time-lapse fluorescence images of maxicells producing YFP-FtsZ* were recorded with a Hamamatsu ORCA 3-CCD camera. Cell lengths were measured with the Object-Image plug-in [30] of the ImageJ software package [31] . The Huygens Professional software package with a correction factor of 0.42 (calculated from the measurement of control spherical particles) was used to deconvolve fluorescence microscope images. Immunolabeling of bacterial cells was performed as described [32] (link), using purified FtsZ (MVC1), FtsA (MVM1) or ZipA (MVC1) antibody as primary antibody and Alexa Fluor-594 labelled goat anti-rabbit IgG (Invitrogen) as secondary antibody. Line profiles of fluorescent signals were analysed using ImageJ [31] .

HCMEC/D3 and 3D vascularized cell sheets were fixed in 4% paraformaldehyde (PFA) (Electron Microscopy Sciences) for 30 min at 4 °C, washed and then blocked with PBS containing 5% goat serum (Invitrogen) and 0.3% Triton-X100 (Bio-Rad) for 1 h at room temperature. Cells morphology and cytoskeleton organization were determined by a direct immunostaining of F-actin with rhodamine labelled phalloidin (10 μg/mL; Sigma-Aldrich). For the 3D microcapillaries network analysis, specific primary antibodies against PECAM-1 (1:100; R&D Systems), VEGFR2 (1:100; Sigma-Aldrich) and Chondroitin Sulfate (NG2; 1:400; BD Biosciences, San Jose, CA, USA) were incubated with whole construct overnight at 4 °C. 3D vascularized constructs were washed and incubated for 2h at room temperature with Alexa Fluor 633-labelled donkey anti-sheep IgG (1:500; Invitrogen) for PECAM-1, Alexa Fluor 594-labelled goat anti-rabbit IgG (1:500; Invitrogen) for VEGFR2 and Alexa Fluor 488-labelled goat anti-mouse IgG (1:500; Invitrogen) for NG2. Nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich). Images were visualized using an LSI 700 confocal microscope with Zeiss Axio Imager (Carl Zeiss Microscopy).