Dmribe microscope

Immunostaining was performed as described in the Supplemental information. Samples were mounted in Dako mounting medium (DAKO) and analysed using an Eclipse Ti microscope (Nikon) equipped with a 100 x objective, a CSU-X1 spinning disk confocal head (Yokogawa), and a Coolsnap HQ2 camera (Roper Scientific Instruments), controlled by the Metamorph 7.7 software. Analysis by epifluorescence microscopy was performed using a DMRIBe microscope (LEICA microsystems) using 380 nm, 470 nm, or 546 nm LED source excitation, equipped with a Cascade 512 camera (Roper Scientific) driven by the Metamorph 7.7 software. Images were analyzed using the Metamorph software. The levels of EspA and EspD secreted at bacterial-cell contact sites were quantified by delimiting an area corresponding to the bacterial microcolony according to DAPI staining. The integrated fluorescence intensity of relative EspA/EspD staining in the corresponding area was measured and expressed as a ratio to that obtained for DAPI staining.

Sampled brains were embedded in OCT (Tissue-Tek, Torrance, CA) and frozen in isopentane at -25°C. 20 μm sagittal sections were cut from frozen brains in a cryostat and fixed in 4% paraformaldehyde for 15 min at 21°C. Serial sections were permeabilized for 60 min in PBS containing 0.25% Triton X-100 and 5% newborn goat serum, prior to incubation with primary antibodies at the following dilutions: anti-GFAP (1:500), anti-PN capsular (1:300), and AlexaFluor 568-IB4 (1:100). Alex-conjugated secondary antibodies and Phalloidin were used at a 1:200 dilution. DAPI was used at a 0.1 mg/ml final concentration. Samples were mounted in DAKO fluorescence mounting medium (DAKO Corp.). Fixed samples were analyzed using Eclipse Ti inverted microscopes (Nikon) equipped with a 60 x objective, a CSUX1-A1 spinning disk confocal head (Yokogawa) and a Coolsnap HQ2 camera (Roper Scientific Instruments), or a CSU1-W1 confocal head (Yokogawa) and an ORCA Flash4 CMOS camera (Hamamatsu) controlled by the Metamorph 7.7 software. For live calcein assays and Ca²⁺ imaging, epifluorescence microscopy was performed using a DMRIBe microscope (LEICA microsystems) using 380 nm, 470 nm, or 546 nm LED source excitation, equipped with a Cascade 512 camera (Roper Scientific) driven by the Metamorph (7.7) software. Images were analyzed using the Metamorph software.