Eclipse ti2 a inverted microscope

Fluorescence images were acquired with a ReScan Confocal instrumentation (RCM 1.1 Visible, Confocal, The Netherlands) equipped with a fixed 50 μm pinhole and combined with a Nikon Eclipse Ti2-A inverted microscope with a motorized Z-stage (DI1500, Nikon). The RCM unit was connected to a Toptica CLE laser with the following excitation modes: 405/488/561/640 nm. Images were taken via an sCMOS camera (PCO edge) using a CFI Plan Apochromat X60 lambda-immersion oil objective (NA 1.4/0.13; Nikon). The instrument was operated by the NIS-Elements software (version 5.11). Images were acquired via a z-stack optical series with a step size of 0.1 microns to cover all structures of interest. To estimate both the total number of cells and the number of binucleated cells present in one cell layer, all images were first segmented using the Otsu thresholding algorithm. Identical brightness and contrast conditions were applied for each data set within one experiment. The total number of cells was obtained using the Fiji plugin “Analyzes particles” with a size of 10 μm (Schindelin et al., 2012 (link)).

Three BUVEC isolates were seeded in 12-well plates with coverslips precoated with fibronectin (1:400, Sigma-Aldrich, F1141-2MG) and infected either with T. gondii Me49 or NED tachyzoites at sub-confluency (MOI 1:2). At 24 h p. i., all samples were fixed in 4% paraformaldehyde (15 min, RT) and washed three times in sterile PBS. The samples were incubated in a blocking/permeabilization solution (PBS with 3% BSA and 0.3% Triton X-100) for 1 h at RT. Thereafter, they were incubated in primary antibody solutions (Table 1) at 4°C in a humidified chamber overnight. The samples were then washed three times with 1X PBS and incubated in secondary antibody solutions (Table 1) for 30 min at RT and darkness. Host cell nuclei were labeled with DAPI present in the mounting medium solution (Fluoromount G-DAPI, Thermo Fisher, cat. Number 495952). The samples were analyzed with ReScan Confocal instrumentation (RCM 1.1 Visible, Confocal.nl) combined with a Nikon Eclipse Ti2-A inverted microscope.