Sp5 x

Capsular polysaccharides type 5 (CP5) or 8 (CP8) were quantified by the immuno-slot-blotting method with rabbit anti-CP5 or anti-CP8 antisera, essentially as described by Luong et al.[24] (link). CP8 production was determined by indirect immunofluorescence. After culture for 8 h in BHI medium at 37°C, bacteria were spotted in diagnostic slides. The slide was incubated with rabbit anti-CP8 antiserum for 1 h at 37°C in humid chamber. After 2 washes, the slide was incubated with secondary antibody, AlexaFluor 647 goat anti-rabbit immunoglobulin G (Invitrogen, Paisley, UK). Total bacteria were labeled with a 1∶1 mixture of vancomycin and vancomycin-Bodipy FL (VBFL) fluorochrome (Invitrogen) at a concentration of 0.8 µg/ml for 15 min. Images were analyzed with confocal microscopy Leica SP5 X (Leica, Solms, Germany) and treated with Leica software: LAS AF Lite (Leica Application Suite 2.6.0).
CP5 or CP8 were quantified by the immuno-slot-blotting method with rabbit anti-CP5 or anti-CP8 antisera, essentially as described by Luong et al.[24] (link). Experimental details are given in the supplementary material (Supplementary Protocol S1).

A confocal laser scanning microscope (Leica SP5-X, Leica Microsystems) equipped with a 63× (numerical aperture 1.2) water-immersion objective was used for all microscopic studies, except super-resolution imaging. A freely tunable white light laser was used for excitation. Unless stated otherwise, the following settings were used: TB, 620 nm excitation, 627–720 nm detection; CFW, 355 nm excitation, 400–460 nm detection; Pontamine Fast Scarlet, 500 nm excitation, 580–650 nm detection; Propidium iodide, 535 nm excitation, 590–660 nm detection. The excitation-emission scans (λ²-scans) were conducted with the following parameters: 470–650 nm excitation range, 19 steps with 10 nm step size, constant laser power, 480–720 nm detection range, 20 nm detection band width, 12 steps with 20 nm step size. The staining intensity of cell wall components was measured by acquiring an image of the powder surface using the exactly same settings for all measurements.
Super-resolution microscopy was performed using a Zeiss Elyra PS1 instrument. TB was excited by a 642 nm laser and emitted light was filtered with a 650 nm long-pass filter. Light was detected on a sCMOS camera (PCO Edge 4.2). A 100× 1.46 NA Zeiss Apochromat oil-immersion objective was used together with a 51 μm grating. Z-stacks were recorded with 3 phase-changes and 3 grating rotations for each section.