The images were captured with Nikon Microphot-Fx and Arkon software and imported to Adobe Photoshop 7 (Adobe System Incorporated, San Jose, CA). Finally, the cropped images were assembled into figures using Canvas 9 (Deneba, Miami, FL). For the flocculation studies, following o.n. growth, the cultures were transferred to test tubes and incubated for 10 min. For scanning electron microscopy (SEM) observations, C. albicans cells were grown in YEPD in the absence or presence of Congo red (50 μg/ml) at 28°C for 2, 6 and 24 h. After centrifuging, the cells were washed twice in distilled

water and fixed with 2.5% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 2% (w/v) sucrose, for 20 min at room temperature (r.t.). After 3 washes in the same buffer, the cells were postfixed with 1% (w/v) OsO4 for 1 h, dehydrated through graded ethanol concentrations, critical point-dried in CO2 (CPD 030 Balzers device, find more Bal-Tec, Balzers) and gold coated by sputtering (SCD 040 Balzers device, Bal-Tec). The samples were examined

with a Cambridge Stereoscan 360 scanning electron microscope (Cambridge Instruments, Cambridge, United Kingdom). For transmission electron microscopy (TEM), cells were prefixed with glutaraldehyde, as previously mentioned, then post-fixed with the OsO4 solution o.n., at 4°C. The cells were then dehydrated in acetone gradient and embedded in epoxy resin (Agar 100 resin, Agar Scientific Ltd, Stansted, UK), as per routine procedures. Selleck Crenigacestat Ultrathin sections, obtained with an LKB ultramicrotome (LKB, Bromma, Sweden), were stained with uranyl acetate and lead citrate. These were examined with a Philips 208 transmission electron microscope (FEI Company, Eindhoven, Netherlands). Immuno-labelling studies in Electron Microscopy Terminal deoxynucleotidyl transferase (EM) For β-glucan localization in the post-embedding procedure, the ultrathin sections, obtained as described

above, and collected on gold grids, were treated for 3 min with 0.5 mg of sodium borohydride per ml of ice-cold distilled water. After being washed in ice-cold distilled water (3 times, for 5 min) and in PBS containing 0.5% (w/v) bovine serum albumin, 0.05% Tween 20, and 5% fetal serum (3 times, 5 min each time), the sections were incubated with mAb 1E12 (diluted 1:10) o.n. at 4°C. After being washed at r.t. for 2 h by floating the grids on drops of PBS, the samples were labeled with rabbit anti-mouse immunoglobulin M (IgM) gold conjugate 10 nm (diluted 1:10; Sigma) and then washed in PBS buffer at r.t for 3 h. For negative control, the sections were incubated with IgM monoclonal antibody or with goat anti-mouse IgG-gold alone. Adhesion to buccal ephitelial cells (BEC) Adhesion to buccal epithelial cells (BEC) was assayed as described previously [28]. Yeast cells were grown for 24 h at 28°C in Winge (0.3% yeast extract, 0.2% glucose), washed twice with PBS (0.02 M NaH2PO4 H2O, 0.02 M YH25448 Na2HPO4 12H2O, 0.15 M NaCl, pH 7.