02% (60:40:9; v/v/v), after development, the plates were dried, soaked in 0.5% polymethacrylate in hexane, dried, and blocked for 2 h with 1% of BSA in PBS. Plates were then incubated with mAb MEST-3 this website overnight followed by sequential incubations with rabbit anti-mouse IgG and 125I-labeled protein A (2 × 107 cpm/50 ml of BSA/PBS). Indirect immunoLY2090314 in vivo fluorescence Fungi were fixed with 1% formaldehyde in PBS for 10 min. Cells were washed, suspended in 1 ml of PBS, and 20 μl of the solution was added to a coverslip pre-treated with poly-L-lysine 0.1% during 1 h. Air
dried preparations were soaked for 1 h in PBS containing 5% of BSA, and incubated subsequently with culture supernatant of mAb MEST-3 (2 h), biotin-conjugated goat anti-mouse IgG (1 h), and with avidin-conjugated fluorescein (1 h). After each incubation
the coverslips were washed five times with PBS. The coverslips were examined with an epifluorescence microscope . Control experiments for each fungus were carried out, in the presence of an irrelevant monoclonal antibody, and no fluorescence was observed. Cell growth To evaluate the influence of mAbs directed to GSLs on the growth of different fungi, yeasts (104/ml) were incubated in 96-well plate in the presence of mAbs MEST-1, -2, or -3 for 24 h at 37°C, in concentration ranging from 2.5 to 50 μg/ml. The growth rate was evaluated by two procedures; 1) viable CFU were evaluated by plating 100 μl of the samples onto BHI or PGY agar plates, followed by incubation Dolichyl-phosphate-mannose-protein mannosyltransferase for 2 days at 37°C, and colony counting; or 2) 5 μl of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Tubastatin A in vivo bromide (MTT) solution (5 mg/ml MTT in phosphate-buffered saline, pH 7,4) were added to each well and the plates were further incubated at 37°C, for 3 h, after incubation the medium containing MTT was partially removed, and dimethyl sulfoxide (100 μl) was added to solubilize the MTT formazan product . The absorbance of each well was measured at 580 nm by a microtiter ELISA plate reader. Control systems were similarly treated with an irrelevant immunoglobulin
(normal mouse total Ig) and plated. All experiments were repeated three times in triplicates, and the results shown are a representative of these experiments. Fungal differentiation – yeast to mycelium 104 viable yeasts were suspended in 1 ml of PGY (P. brasiliensis) or BHI (H. capsulatum and S. schenckii) medium. The suspension was incubated in a 24-well plate and supplemented with mAb MEST-1, -2, or -3 (at a concentration of 2.5, 10, 25 or 50 μg/ml), after one hour at 37°C, 24-well plate was transferred to a 24°C incubator and kept for 2 days. The number of yeast showing hyphae growth was counted, and presented as percentage of those incubated with irrelevant immunoglobulins (normal mouse total Ig). In control experiment 100% of yeast showed hyphae formation.