Cell fixation occurred 16 h following transfection with 4% paraformaldehyde in phosphate-buffered saline, pH 7.4 (PBS) for 15 min. Slides were frozen at −40 °C until analyzed by immunocytochemistry. Slide wells were blocked with PBS with 1% bovine serum albumin (BSA) weight by volume (w/v). Three brief washes were performed between each step using PBS. Wells were probed with mouse monoclonal antibody 2A10 anti-Plasmodium falciparum CS repeat obtained through BEI Resources (NIAID, NIH: Monoclonal Antibody 2A10 Anti-Plasmodium falciparum Circumsporozoite Protein produced in vitro, MRA-183A, contributed by Elizabeth Nardin) diluted in PBS with 1% BSA to 10 µg/mL. Secondary detection was accomplished with 5 µg/mL fluorescein isothiocyanate (FITC) conjugated Goat anti-Mouse IgG (Southern Biotech 1034-02) supplemented with 100 µg/mL of 4′,6-diamidino-2-phenylindole (DAPI) in PBS with 1% BSA. Following final washes, slides were allowed to air dry under reduced light, then mounted using Fluoromount-G (Southern Biotech 0100-01). Images were acquired using a BX53F microscope and DP80 camera (Olympus). Microscope settings (exposure time, bulb intensity) were consistent across all images within an experiment. Images were acquired at 63× magnification. For fluorescence area detection, the FITC and DAPI fluorescent areas (µm2) were measured across twenty fields at ×25.2 magnification for each condition using Olympus cellSens imaging software. To account for the varied size and quantity of cells between fields, FITC measurements were normalized to the DAPI measurements within the same field, resulting in a FITC/DAPI area ratio. Background levels of this ratio were detected under mock conditions where the cells were only exposed to the transfection reagents, not the mRNA. Commercially obtained antibodies were used according to manufacturer’s instructions.

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