Flowview fv1000 laser scanning confocal imaging system

Mature schizont-stage parasites were purified from whole-blood samples from vivax malaria patients. The parasite antigen slides were prepared with ice-cold acetone for fixation and blocked with PBS containing 5% skim milk at 37 °C for 30 min. The following primary antibodies were used: rabbit anti-PvRBP1a-34 (1:50) and anti-PvRBP1b-32 (1:50) with anti-PvRAMA (1:200), anti-PvDBP-RII (1:100) and anti-PvMSP1-19 (1:200). The following secondary antibodies were used: Alexa 546-conjugated goat anti-mouse IgG or Alexa 488-conjugated goat anti-rabbit IgG (Invitrogen) at a 1:500 dilution. DAPI (4′,6′-diamidino-2-phenylindole, Invitrogen) was applied at a 1:1,000 dilution at 37 °C for 30 min to stain the nuclei. The slides were mounted with ProLong Gold antifade reagent (Invitrogen) and visualized under oil immersion using a Flowview^® 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 and prepared for publication with Adobe Photoshop CS5 (Adobe Systems, San Jose, CA, USA). Each fluorescence graphic contained more than 500 pixels calculated by Image J (NIH, USA).

Surface expression of each PvGAMA fragment on HEK 293T cells transfected with target plasmid DNA was detected using a 1:100 dilution of rabbit antisera against PvGAMA-Ecto or rabbit antisera against PvDBPII as the primary antibody and Alexa Fluor 568-conjugated goat anti-rabbit antibodies (Invitrogen) as the secondary antibody. The GFP-tag of each recombinant protein was detected using a 488-nm laser. The cells were observed unfixed on a Flowview^® FV1000 Laser Scanning Confocal Imaging System (Olympus, Tokyo, Japan). The images were analysed and manipulated using Adobe Photoshop CS5.