IFA was performed essentially according to [70 (link)]. Where iRBCs were settled onto a poly-L-lysine (Sigma, P8920) coated coverslip and fixed with 4% paraformaldehyde/0.0075% glutaraldehyde. Following fixation, the cells were permeabilised with 0.1 M glycine/0.1% Triton X-100 for 12 minutes at RT. Alternatively, thin blood smears of parasites were fixed in 90% acetone/10% methanol for 5 mins where specified. Coverslips or slides were blocked with 3% BSA/0.02% Triton X-100/1x PBS and probed overnight at 4°C, with rabbit anti-Nluc (12.5 μg/mL), mouse anti-EXP2 (10 μg/mL), rabbit anti-ERC (1:1000), mouse anti-FLAG M2 (Sigma, 10 μg/mL), mouse anti-HA (Sigma clone HA-7; 1:500), rabbit anti-SBP1 (1:500) [36 (link)] and rabbit anti-STEVOR (PF3D7_1254100, 1:500) [71 (link)]. After washing goat anti-rabbit Alexa Fluor 594 and Goat anti-mouse Alexa Fluor 488 (1:2000) secondary antibodies were applied for one hour at RT. Fixed material was mounted in VECTASHIELD with DAPI and imaged on Zeiss Cell Axio Observer (Carl Zeiss). Image acquisition was performed with Zen Blue imaging software.
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