Zen 3.1 blue software

Formalin-fixed paraffin embedded (FFPE) bone slices of the right pelvis of the infected mouse and of the non-infected control mouse were de-paraffinised using xylene (Carl Roth) and hydrated in a series of graded alcohols. Briefly, slides were placed 2× in xylene, 1× in xylene:ethanol (1:1), 2× in 100% ethanol, and subsequently in 95% ethanol, 70% ethanol, and 50% ethanol followed by rinsing with tap water. The slides were left in each solution for 3 min before proceeding to the fresh next solution. Subsequently, bone slices were either stained with hematoxylin and eosin (Mayer’s hemalum solution and Eosin G 0.1%, both Merck, Darmstadt, Germany) to reveal the tissue structure, or Gram staining was performed according to the manufacture’s protocol (Carl Roth, Karlsruhe, Germany) to visualise bacteria.
Bright field images were acquired using an Axio Observer.Z1 (Carl Zeiss, Jena, Germany) equipped with an AxioCam MR R3 camera (Carl Zeiss) and a Plan Apochromat 40×/0.95 objective or a Plan-Neofluar 63×/1.3 oil immersion objective. The whole slices were scanned in tile-scan mode and stitched using the Zen 3.1 blue software (Carl Zeiss).

Confocal immunofluorescence microscopy experiments were accomplished as previously described by us [48 (link)]. Briefly, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min before being permeabilized through 5 minute-treatment with 0.1% Triton X-100. Cells were then washed with PBS (pH 7.2) and blocked with 0.5% BSA in PBS before being probed with primary antibodies and subsequent secondary fluorophore-conjugated antiserum (Alexa Fluor 488 and 564). Anti-FLAG antibody (M2, Sigma) was used at 1:1000, anti-FLAG polyclonal antibody; 1:400, anti-Myc 9E10; 1:100, anti-Sec31A; 1:100, anti-ERGIC-53; 1:100, anti-GM130; 1:100, anti-EEA1; 1:100, anti-Sec16A; 1:200, and anti-MAVS; 1:100. Secondary fluorophore-conjugated antiserum (Alexa Fluor 488 and 564) was used at 1:500 in PBS 0.5% BSA. The nucleus was labeled by 4′,6-diamidino-2-phenylindole (DAPI) staining. The confocal micrographs represent a single optical section (Z-stack) of cells. Images were acquired from a LSM 510 inverted microscope (Zeiss) combined to LSM v3.2 software (Zeiss). Colocalization of labeled protein was assessed by linescan analysis using “Profile” function in the ZEN 3.1 blue software (Zeiss). The pixel intensity in each channel is measured along a line drawn on the image and is plotted versus distance along the line.