Eight well microscopy μ slide

hCMEC/D3 cells were seeded until confluent in eight‐well microscopy μ‐slide (ibidi, cat no. 80826). M. marinum E11 and M. marinumeccCb1::tn were grown until the exponential growth phase, spun down, and resuspended in specialised medium. Brain endothelial cells were infected with an MOI of 10 or 50, incubated for 2 or 24 hr, washed with PBS and fixated with 4% PFA dissolved in PBS for 20 min. For labelling, cells were blocked for 60 min in block buffer (5% NGS in 0.3% Triton X100). Samples were incubated with anti‐LAMP1 (Cell Signalling, cat. no 9091P, 1:100) in antibody buffer (1% BSA and 0.3% Triton X100 in PBS) overnight at 4 °C. Samples were washed with PBS and incubated with Alexa‐Fluor‐488 goat‐anti‐rabbit (Molecular Probes, cat. no A‐11008, 1:400) in antibody buffer for 90 min at RT. Cells were washed with PBS, incubated with Hoechst (1:1000, Molecular Probes, cat. no 33258) for 1 min, washed with PBS, and stored in PBS at 4 °C until further analysis with confocal microscopy (Leica TCS SP8 X Confocal Microscope). Leica Application Suite X software was used for 3D analysis.

Bacterial infection was monitored initially with a Leica MZ16FA fluorescence microscope. Bright field and fluorescence images were generated with a Leica DFC420C camera. Early granuloma formation was analysed visually and quantified with custom‐made pixel‐counting software (http://www.elaborant.com). Confocal analysis was performed on hCMEC/D3 cells and larvae, embedded in 1% low melting‐point agarose (Boehringer Mannheim, 12841221‐01) in an eight‐well microscopy μ‐slide (ibidi). Analysis was performed with a confocal laser‐scanning microscope (Leica TCS SP8 X Confocal Microscope). Leica Application Suite X software and ImageJ software were used to adjust brightness and contrast and to create overlay images and 3D models.