Fluorescence Microscopy and TEM Analysis of Candida Albicans Cell Wall

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.