Immunoelectron microscopy of saponin permeabilized IE to confirm PfMC-2TM presence at the erythrocyte membrane. a, b A pre-embedding staining protocol was applied to analyse PfMC-2TM membrane association by immunoelectron microscopy. Trophozoite IE were permeabilized with saponin and incubated with the immune serum rabbit α-PfMC-2TM-CT (I) or the respective pre-immune serum (PI). Recognized proteins are visualized with 10 nm gold particles. Different sections are shown depicting PfMC-2TM association with the erythrocyte membrane (a) and with Maurer’s clefts (b). c 20 randomly selected infected erythrocytes were quantified for their gold particle localizations, which are divided into the sections total cell (total), erythrocyte membrane (EM), Maurer’s clefts (MC), parasite membrane/parasitophorous vacuole membrane (PM/PVM) and other localization (others). Data are presented as mean ± SEM. Statistical analyses were done with an unpaired t test. Significant differences between α-PfMC-2TM-CT and pre-immune serum were observed for total cells (p = 0.0003), an erythrocyte membrane association (p = 0.002) and labelling of the Maurer’s clefts (p = 0.013).