Dapi fluormount g

For immunofluorescence analysis, HFF monolayers infected with Toxoplasma parasites were grown on coverslips and fixed at the indicated time points in 4% paraformaldehyde for 20 min at RT. Afterwards coverslips were permeabilised in 0.2% Triton X–100 in 1 × PBS for 20 min, followed by blocking (3% BSA and 0.2% Triton X–100 in 1x PBS) for at least 30 min. The staining was performed using indicated combinations of primary antibodies (Supplementary file 1) for 1 hr and followed by secondary Alexa Fluor 488 or Alexa Fluor 594 conjugated antibodies (1 ∶ 3000, Invitrogen – Molecular Probes) for another 45 min. Nuclei were stained with DAPI-Fluormount-G (SouthernBiotech).

The cells were fixed in 4% paraformaldehyde for 15 min and incubated in 1% Triton X-100 for 15 min at 25 °C. The cells were then incubated with the primary antibody, E-cadherin (1:500) or α-smooth muscle actin (α-SMA) (1:500), overnight at 4 °C, followed by incubation with the secondary antibodies, Alexa Fluor 488 goat anti-rabbit and Alexa Fluor 546 goat anti-mouse, for 1 h at 25 °C. The coverslips were mounted in DAPI Fluormount-G (SouthernBiotech, Birmingham, AL) and scanned with laser-scanning confocal microscopy (FLUOVIEW FV1000, Olympus, Central Valley, PA).

For the monocyte infiltration into atherosclerotic lesion assay, experimental male mice were fed a WD for 10 weeks. Clodronate-liposomes (250 μl, Liposoma) were i.v. injected in order to transiently deplete monocytes, followed by i.v. injection of 250 μl fluorescent microspheres 48 h later. Fluoresbrite FITC-dyed (YG, 0.5 μm) plain microspheres (2.5% solids [w/v]; Polysciences) were diluted 1:25 in PBS^{26 (link),27 (link)}. Mice were euthanized and hearts with aortic root was then used for consecutive sections from the atrioventricular valve at a thickness of 20 μm. Nuclei were counter-stained by DAPI Fluor mount-G (SouthernBiotech). Images were then captured using a fluorescence microscope (Carl Zeiss). Beads that reflect monocyte recruitment were quantified in 3–5 aortic sinus sections per mouse.
For the murine peritoneal mono-macrophage recruitment, 1 ml of sterile 4% thioglycolate media was injected intra-peritoneally. The cells from murine peritoneal cavities were harvested 3 days later, and analyzed by cell counter or flow cytometry.

HEK293T cells were grown on 0,01% poly-L-lysine coated coverslips and analyzed 24 hours post-transfection with DNAs coding: IFITMs, HIV-1 Gag-Pol plus non-structural viral proteins, NL4-3 Env, a miniviral genome coding CD8, as well as a small amount of Gag-GFP (ratio 3:1:1:1:0,1). After Formalin fixation, cells were incubated with the following antibodies: anti-Flag (F7425, Sigma), followed by DyLight 649- conjugated sheep, or FITC-conjugated goat anti-rabbit IgG (STAR36D649 AbD serotec and FI-1000 Vector). DAPI-containing mounting medium was finally used (DAPI Fluormount G, Southern biotech). Images were acquired using a spectral Leica sp5 and analyzed with the Fiji software [54 (link)]. Two-dimensional graphs representing pixel intensities (gray level) were plotted along a 30-μm lines (yellow on Figure 3B), using Plot Profile tool in Image-J. The extent of reciprocal co-localization between Gag and IFITMs was quantified using the Manders overlap coefficient (Fuji image software) on more than 40 cells per condition.

Thin blood films were made on glass slides and fixed in 4% paraformaldehyde in PBS for 20 min. The slides were then permeabilised with 0.1% Triton-X/PBS for 10 min, washed and blocked overnight in 4% BSA/PBS. Antigens were labelled with suitable primary and secondary antibodies in 4% BSA/PBS with 5 min PBS washes in between. Slides were finally air dried and mounted with DAPI-Fluormount-G (SouthernBiotech).
Staining of the RON4 junction in CB-EME expressing was performed by fixation and immunostaining in solution as described previously (Riglar et al., 2011 (link)).
For image acquisition, z–stacks were collected using a UPLSAPO 100 × oil (1.40NA) objective on a Deltavision Core microscope (Image Solutions – Applied Precision, GE) attached to a CoolSNAP HQ2 CCD camera. Deconvolution was performed using SoftWoRx Suite 2.0 (Applied Precision, GE).
An Elyra S1 microscope with Superresolution Structured Illumination (SR-SIM) (Zeiss) was used for super-resolution imaging.
Colocalisation analysis was performed by using the Coloc two plugin in ImageJ and obtaining the Pearson´s R value for two defined channels.