Thimerosal

For immunofluorescence imaging, the BBB monoculture comprising hCMEC/D3 alone was cultured on the membranes of the static and the dynamic model. At days 3, 5 and 8, the membranes were cut and placed on a microscopic slide and subsequently fixed, blocked, and permeabilized using 4% paraformaldehyde (Sigma), and 0.01 M PBS (pH 7.4) supplemented with 0.05% thimerosal (Sigma), 10% normal horse serum, and 1% Triton X-100 (Sigma), respectively. Next, the cells were stained overnight using the following primary antibodies: a mouse anti-human−1 antibody (1/100) (BD Pharmingen, Erembodegem, Belgium). Next, cells were stained with a secondary fluorescein isothiocyanate (FITC)-labeled donkey anti-mouse antibody (1/100) (Jackson ImmunoResearch, Newmarket, UK) for 2 h at room temperature. Finally, cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma), and the membrane containing the cells was mounted in citifluor (Citifluor Ltd., London, UK) following its careful removal from the silicon casket of TripleB membranes and the transwell insert, and was subsequently stored at 4 °C. The images were taken using an UltraVIEW confocal microscope system (Perkin Elmer, Waltham, MA, USA). The analysis of the images was performed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Wild-type C. albicans SC5314 was maintained as previously described (Leonhardt et al., 2015 (link)). For experiments, colonies were transferred to M199 medium (PAA), pH 4 and cultured at 30°C to stationary phase. Germ tubes were induced by culturing in M199 medium, pH 8 for 1 h at 37°C. Germ tubes were inactivated by washing them in phosphate-buffered saline (PBS) and followed by incubation in PBS containing 0.1% thimerosal (Sigma-Aldrich) for 1 h at 37°C with shaking. Afterwards, germ tubes were washed five times and then re-suspended in RPMI-1640. Killing was confirmed by plate counts on YPD (2% D-glucose, 1% peptone, 5% yeast extract, Roth) agar plates.

For fluorescence microscopy, cells were fixed in 50 mM thimerosal (Sigma-Aldrich) and stained for β-glucan (1.5 µg/ml Fc-Dectin-1 plus anti-human IgG conjugated to Alexa Fluor 488; green), chitin (50 µg/ml wheat germ agglutinin conjugated to Alexa Fluor 350; blue), and mannan (25 µg/ml concanavalin A conjugated to Texas Red; red). All samples were examined by phase differential interference contrast (DIC) and fluorescence microscopy using a Zeiss Axioplan 2 microscope. Images were recorded digitally using the Openlab system (Openlab v 4.04: Improvision, Coventry, UK) with a Hamamatsu C4742- 95 digital camera (Hamamatsu Photonics, Hamamatsu, Japan).
High-pressure freeze substitution transmission electron microscopy on normoxic and hypoxic C. albicans cells was performed as described previously (103 (link), 104 (link)), cutting ultrathin sections of 100 nm in thickness. Samples were imaged with a Philips CM10 transmission microscope (FEI, United Kingdom) equipped with a Gatan Bioscan 792 camera, and the images were recorded using a Digital Micrograph (Gatan, Abingdon Oxon, United Kingdom). The thicknesses of the inner chitin-glucan and outer mannan layers of the cell wall were measured by averaging >30 measurements for each cell (n > 30 cells) using ImageJ.