9100 02 emccd camera

Transfected cells were processed for immunofluorescence as described^{50 (link)}. Briefly, cells were fixed for 10 min with 3% paraformaldehyde at room temperature, permeabilized with 0.1% Triton-X100 in PBS, incubated with primary antibodies, washed, incubated with secondary antibodies, and mounted with ProLong Gold antifade mounting solution (Thermo Fisher Scientific). Cells were observed with epifluorescence microscopes: Zeiss AxioImager M2m equipped with AxioCam color CCD camera, with Plan-Neofluar 40× lens (NA 0.75) and Plan-Apochromat 63× lens (NA 1.4). Confocal images were acquired at a Perkin Elmer UltraVIEW spinning disk confocal microscope with EM-CCD camera and Plan-Apochromat 63× lens (NA 1.4); or at a Leica TCS SP5 or TCS SP8 SMD FLIM laser scanning confocal microscope equipped with HC PLAPO CS2 63x lens (NA 1.4). For quantitative analysis of the projected cell area, transfected cells were randomly imaged at a wide field microscope (Zeiss Axio Observer.Z1 equipped with Hamamatsu 9100 - 02 EM CCD Camera). For evaluation of the subcellular localization of transfected and of endogenous proteins, confocal images were visually analyzed. For quantification, 2–4 independent experiments per condition were analyzed using Fiji^{51 (link)}.

Intravital microscopy was performed in the bifurcation of the left carotid artery by means of epifluorescence microscopy. The right jugular vein was canulated with a catheter for antibody and dye injection. After exposure of the left carotid artery, antibodies (1 μg) to CD11b (M1/70, Biolegend), Ly6C (HK1.4, Biolegend) and Ly6G (1A8, Biolegend) were sequentially administered to label myeloid cells, neutrophils and classical monocytes, respectively. Recordings were made 3 min after injection of each antibody. The diameters of the external carotid arteries were 268.8 ± 34.4 µm in the control group and 267.4 ± 37.1 µm in the endothelial ACKR3 deficient group. Intravital microscopy was performed using an Olympus BX51 microscope equipped with a Hamamatsu 9100-02 EMCCD camera and a ×10 saline-immersion objective. For image acquisition and analysis, Olympus Cell-R software was used.

PBS with 1% agarose was used to make a pad on a microscope slide. 10 µL sample with an OD₆₀₀ of 20 was placed on the pad and covered with a cover slip. Microscope images were collected using separate channels for phase contrast and fluorescence in a Zeiss Axio Imager.Z1 microscope equipped with X-Cite 120 Illumination (EXFO Photonic Solutions Inc.) and a 9100-02 EM-CCD camera (Hamamatsu Photonics). Data was handled with the Zen microscopy software and images analyzed using Fiji (ImageJ) [21 (link)].

Gelatin degradation was detected as published^{27 (link),52 (link)}. Glass coverslips coated for 1 h at room T with 0.5 mg/ml poly-L-lysine (Sigma-Aldrich) were quenched 15 min at 4 °C with 0.5% glutaraldehyde in PBS, and then coated for 10 min at room T with Oregon–green–conjugated gelatin (Life Technologies) diluted 1:4 in 0.2% gelatin in PBS. Subsequently the coverslips were additionally coated with 10 μg/ml fibronectin in PBS for 1 h at 37 °C. Cells were plated on gelatin-coated coverslips for 5 h before fixation and immunostaining. Gelatin degradation was detected at a Zeiss Axio Observer.Z1 equipped with Hamamatsu 9100 - 02 EM CCD Camera and Plan-Apochromat 63x (NA 1.4) lens. The dark areas of gelatin degradation and the projected cell areas were quantified by ImageJ on thresholded images. Data were pooled from 2 to 3 independent experiments.