To detect IgM binding to merozoites, parasites were tightly synchronized before late stage schizonts were purified using 70% Percoll, put back into culture containing RPMI + Albumax II with the addition of 20% human serum or 0.125 mg/ml purified human IgM (Sigma). IgM was detected using an Alexa Fluor 488 goat anti-human IgM μ chain antibody (Molecular Probes; preadsorbed against human IgG) at 1:1000. The slides were viewed on a Zeiss Axioplan 2 imaging system with Plan Apochromat 100×/1.4 oil immersion objective. Images were captured using Axiovision 4.6.3 software and edited using Adobe Photoshop.
For immunogold labeling, merozoites were fixed in 4% paraformaldehyde in 0.1m phosphate buffer at pH 7.4 for 1 h at room temperature, rinsed three times in buffer, and infiltrated with 1% and then 10% gelatin before immersing in 2.3 m sucrose in phosphate buffer overnight at 4 °C for cryoprotection. Frozen samples were prepared by mounting onto aluminum pins and rapidly immersing in liquid nitrogen in preparation for ultrathin 80 nm sectioning on a Leica EM FC6 ultramicrotome. Ultra thin sections were labeled as per Tokuyasu (62 ), with a rabbit anti-human IgM antiserum (Abcam) diluted 1:25, and detected with 10-nm protein A gold. Imaging was performed on an FEI 120kV Spirit Biotwin with a Tietz F4.15 CCD camera.
For immunogold labeling, merozoites were fixed in 4% paraformaldehyde in 0.1