Immunolocalization of FP-2 by confocal microscopy. Erythrocytes infected with P. falciparum trophozoites were smeared on a glass slide, fixed with ice-cold methanol, and incubated with polyclonal FP-2 (A) or SERP (B) antibodies followed by Alexa 488 (green) or Alexa 594 (red) conjugated goat anti-rabbit IgG. Merged images of differential interference contrast (DIC) and fluorescence microscopy are shown on the right. C and D, slides were incubated with polyclonal FP-2 together with either plasmepsin IV (C) antibodies followed by Alexa-488-conjugated (green) goat anti-rabbit IgG and Alexa 594-conjugated (red) goat anti-mouse IgG. Merged images of green and red micrographs are shown on the right. The FP-2 antibody stained the entire trophozoite and vesicles or vesicle-like structures of unknown identity were seen extending into the erythrocyte cytoplasm. Co-staining of infected erythrocytes with antibodies to plasmepsin IV, which is present exclusively in the food, confirmed that FP-2 is present in the food vacuole as well as in the area that lies outside of the food vacuole (C). A comparison of FP-2 and SERP staining shows that the FP-2 is localized in the PVM as well as in a compartment that is distinct from the PVM.Dhawan S, Dua M, Chishti AH, Hanspal M. Ankyrin peptide blocks falcipain-2-mediated malaria parasite release from red blood cells. J Biol Chem. 2003 278:30180-6

Biotin localizes to the parasitophorous vacuole. Trophozoite-infected erythrocytes were permeabilized with SLO, biotin-labeled, and spread onto microscope slides. Cells were fixed and incubated with antibodies to SERP (A), a vacuolar resident protein. Conjugated secondary antibodies (green color) co-localize with Cy3-conjugated streptavidin (red color). B and C show dual labeling of biotin and the erythrocyte membrane protein band 3 (B) or of biotin and the parasite cytosolic protein aldolase (C). Cells were imaged using a laser scanning confocal microscope. The left panels (TM/nomarski) show the transmission images of the respective fields, and the right panels (merge) show an overlay of fluorescence images for each of the antibodies used. The images show a distinct ring around the periphery of the intracellular parasite. The biotin co-localizes with the vacuolar marker protein SERP (A). This location is distinct from the location of the erythrocyte membrane protein band 3 and from the location of the parasite cytosolic protein aldolase (B and C). A ring-like staining around the periphery of the parasite can be attributable to a reaction of the biotin with membrane proteins of the PVM and with soluble vacuolar proteins.Nyalwidhe J, Baumeister S, Hibbs AR, Tawill S, Papakrivos J, Volker U, Lingelbach K. A nonpermeant biotin derivative gains access to the parasitophorous vacuole in Plasmodium falciparum-infected erythrocytes permeabilized with streptolysin O. J Biol Chem. 2002 277:40005-11.