Light editions of zen 2012 and 2009

Purified extracellular merozoites and sporozoites were transferred to poly-L-lysine treated glass slides (Polysciences Inc., Hirschberg an der Bergstrasse, Germany) and air dried before fixation. Parasites were either fixed with 4% paraformaldehyde in PBS for 10 min followed by permeabilization with 0.25% TritonX-100 in PBS for 10 min or fixed in ice-cold 100% methanol for 10 min and then blocked with 4% BSA in PBS for 2 h at room temperature. A 1:50 dilution of chicken anti-rCSUI_005805 polyclonal sera was added and incubated for 2 h at room temperature followed by 1 h incubation with a 1:300 dilution of Alexa Fluor^® (A488) goat anti-chicken IgG (Invitrogen, Eugene, OR, USA). Nuclei were visualized by staining with 1 μg/ml of 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min prior to mounting under coverslips with Aqua-Poly/Mount (Polysciences Inc.). The slides were washed five times with PBS for 25 min after each step described above. Imaging was done on a Zeiss Axio Imager Z2 wide-field fluorescence microscope (×63 oil immersion objective) and a Zeiss LSM 510 Meta-confocal laser scanning microscope (×63 oil immersion objective). Images were analyzed with Light Editions of Zen 2012 and 2009 (Carl Zeiss Microimaging GmbH, Jena, Germany).

Merozoites and gamonts from cell culture supernatants were washed once with PBS at room temperature and transferred to poly-L-lysine treated glass slides (Polysciences Inc., Hirschberg an der Bergstrasse, Germany) and air dried before fixation. Parasites were either fixed with 4% paraformaldehyde in PBS for 10 min followed by permeabilization with 0.25% TritonX-100 in PBS for 10 min or fixed in ice-cold 100% methanol for 10 min and then blocked with 4% bovine serum albumin (Sigma-Aldrich, St. Louis, Missouri, USA) in PBS for 2 h at room temperature. A 1:500 dilution of anti-rCSUI_001473 polyclonal sera was added and incubated for 2 h at room temperature followed by 1 h incubation with a 1:300 dilution of Alexa Fluor® (A488) goat anti-chicken IgY (Invitrogen, Eugene, OR, USA). The slides were washed five times with PBS for 25 min after each step described above. 4′,6-diamidino-2-phenylindole, DAPI (5 μg/ml) was included in the Fluoromount-G® mounting medium (Thermo Fischer Scientific) for nuclear staining. Imaging was carried out with a Zeiss LSM 510 Meta-confocal laser scanning microscope (× 63 oil immersion objective). Images were analyzed with Light Editions of Zen 2012 and 2009 (Carl Zeiss Microimaging GmbH, Jena, Germany).