Fluoview fv1000 laser scanning confocal imaging system

P. vivax thin smears from Korean isolates schizonts and P. knowlesi A1-H.1 schizonts were prepared with ice cold acetone fixation, and the smears were blocked with 5% BSA in PBS for 30 minutes at 37 °C. The parasite smears were incubated with a 1:100 dilution of mice monoclonal antibodies and anti-PvMSP1-19 antibody from rabbit for 1 hour at 37 °C, followed by incubation with a 1:500 dilution of Alexa Fluor 488 conjugated goat anti-mouse IgG (Invitrogen) and Alexa Fluor 546 conjugated goat anti-rabbit IgG (Invitrogen) as secondary antibodies for 30 minutes at 37 °C. The parasite nuclei were stained with 2 μg/ml DAPI (4′,6-diamidino-2-phenylindole) in the secondary antibody mixture. Slides were mounted by ProLong^® Gold Antifade reagent (Invitrogen) and visualized by a Fluoview^® FV1000 Laser Scanning Confocal Imaging System (Olympus, Tokyo, Japan) under the 60x objective oil-immersion lens. Images were visualized by FV10-ASW 3.0 viewer software.

To confirm the localization of PvMSP1-19 and PkMSP1P-19, blood smears for indirect immunofluorescence assay (IFA) were prepared as described previously (Li et al., 2012 (link)). Slides smeared with P. knowlesi schizont-enriched blood were fixed with 4% paraformaldehyde and blocked with 5% skim milk in PBS. Rabbit anti-PkMSP1-19 and mouse anti-PkMSP1P-19 diluted in 1:50 were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rabbit IgG or Alexa Fluor 546-conjugated goat anti-mouse IgG secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA) and 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen Life Technologies) were used for secondary antibody staining. The slides were mounted with ProLong Gold Antifade reagent (Invitrogen) and visualized under immersion oil using the FluoView^® FV1000 Laser Scanning Confocal Imaging System (Olympus, Tokyo, Japan) equipped with a 60× oil objective. Images were captured using the FV10-ASW 3.0 Viewer software (Olympus). The fluorescence graphic containing more than 500 pixels was calculated by ImageJ (NIH, Rockville, MD).