Silica gel blue

The schizont stage-rich parasites obtained from short-term in vitro culture were spotted onto multi-well slides and fixed with ice-cold acetone for 3 min, dried, and stored at − 80 °C. Before use, the slides were thawed on silica gel blue (Samchun Chemical, Pyeongtaek, Gyeonggi, ROK) and blocked with PBS containing 5% non-fat milk at 37 °C for 30 min. The slides were incubated with 1:200 diluted primary antibodies (mouse anti-PvMSP1-19 and rabbit anti-Pv32) at 37 °C for 1 h. The PvMSP1-19 was used as the merozoite surface protein marker. After the primary antibody reactions, the slides were stained with Alexa 546-conjugated goat anti-rabbit IgG secondary antibody (Ab) or Alexa 488-conjugated goat anti-mouse IgG secondary Ab (Invitrogen Corp., Carlsbad, CA, USA), and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen Corp.) at 37 °C for 30 min. The slides were mounted with ProLong Gold antifade reagent (Invitrogen Corp.) and viewed under oil immersion with a confocal laser scanning FV200 microscope (Olympus, Tokyo, Japan) equipped with 20× dry and 60× oil objectives. Images were captured with FV10-ASW 3.0 viewer software and prepared for publication with Adobe Photoshop CS5 (Adobe Systems, San Jose, CA, USA).

The enriched schizont-stage parasites were purified by Percoll gradient centrifugation, spotted onto a multi-well slide, fixed in ice-cold acetone for 3 min, air-dried, and stored at -80 °C. Before use, the slides were thawed on silica gel blue (Samchun Chemical, Pyeongtaek, Gyeonggi, ROK) and blocked with PBS containing 5% non-fat milk at 37 °C for 30 min. Next, the slides were incubated with 1:200 diluted primary antibodies, mouse anti-MSP1-19 [15 (link)] and rabbit anti-Pv50, at 37 °C for 1 h. After the reaction, the slides were stained with Alexa 546-conjugated goat anti-rabbit IgG secondary antibody (Ab) or Alexa 488-conjugated goat anti-mouse IgG secondary Ab (Invitrogen, Carlsbad, CA, USA) and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) at 37 °C for 30 min. The slides were mounted with cover slips using mounting medium ProLong Gold antifade reagent (Invitrogen) and observed under oil immersion in a confocal laser scanning FV200 microscope (Olympus, Tokyo, Japan) equipped with 20× dry and 60× oil objectives. Images were visualized with FV10-ASW v.3.0 viewer software, the overlap coefficient analyzed with Image J (Softonic International, Barcelona, Barcelona, SA), and adjusted for publication with Adobe Photoshop CS5 (Adobe Systems, San Jose, CA, USA).