Inverted microscope model ix70

Cells to be photographed were grown in 24-well microtiter plates, washed thrice with phosphate buffered saline (PBS), fixed in 4% formaldehyde in PBS for 60 min, washed thrice, and stored at 4 °C in PBS. Fixed fungal cells were pre-incubated for 30 to 60 min with in LDB2 + 5% BSA at 23 °C. Liposomal stocks were freshly diluted approximately 100-fold into LDB2 + 5% BSA on the same day they were to be used and then incubating with cells such that the DCS12 or DCS78 or BSA protein concentration was approximately 1.0 µg/100 µL. After 1 h incubation at 23 °C, unbound liposomes were washed out with 4 changes of LDB2 + 5% BSA. Ten separate fluorescent images of rhodamine red fluorescent liposomes bound to cells grown on 24 well microtiter plates were taken at 10× or 20× magnification on an Olympus inverted microscope (Model IX70) with a digital camera. The relative area of red fluorescent liposome binding from 10 random images was quantified by taking an 8-bit grey-scale copies of the unmodified red fluorescent JPEG images into Image J (imagej.nih.gov/ij) as described previously for Dectin-2 coated liposomes [3 ]. The green channel of bright field images showing fungal cells and the red fluorescent images of liposomes were merged in Photoshop and enhanced for image presentation.

The ICC protocol was performed as previously described [10 (link)]. In brief, cells were fixed with 2% paraformaldehyde (PFA) in PBS, pH 7.4, for 20 min. Afterwards, the cells were permeabilized with 0.2% Triton X-100 for 20 min, blocked with 1% BSA solution for 45 min, and incubated with primary antibody (1:1000 µg/ml) overnight at the incubator. Upon 3 h of incubation at room temperature with the secondary antibody (1:1000 µg/ml), cells were stained with DAPI (1:1000µg/ml) for 5 min at room temperature. Cells were washed three times in PBS with a pH of 7.4 in between each step. Cells may then be examined using a fluorescent microscope (Olympus, Japan, Inverted Microscope Model IX70). Importantly, identical exposure settings were used to get the pictures. Using Image J software (version 1.50i, National Institute of Health, Bethesda, MD, USA), pictures were also analyzed.