A. tage dependent co-localization of PfHsp70-x and PfEMP1. Upper infected cell (ring stage) shows a higher degree of signal colocalization than lower infected cell (trophozoite). B. ndirect immuno-fluorescence of PfHsp70-x and PfEMP1 using confocal microscopy. In merge and overlay: Green, α-70x; Red, α-ATS; Blue, Hoechst. C. ndirect immuno-fluorescence localization of PfHsp70-x using anti-SBP1, anti-MAHRP2 and anti-STEVOR (STV) antisera in cells infected with 3D7GFP:70x. In merge and overlay images: Green, GFP; red, specific antisera; Blue, Hoechst. No co-localization can be observed between the GFP and antibody-specific signals.Külzer S, Charnaud S, Dagan T, Riedel J, Mandal P, Pesce ER, Blatch GL, Crabb BS, Gilson PR, Przyborski JM. Plasmodium falciparum-encoded exported hsp70/hsp40 chaperone/co-chaperone complexes within the host erythrocyte. Cell Microbiol. 2012 14(11):1784-95

Location of native STEVOR at the iRBC surface. (A) Immunofluorescence assay of live 5A-infected erythrocytes with anti-S1 (upper row) and anti-S2 (middle row) antisera recognizing native STEVOR at the iRBC surface. Either anti-S1 or anti-S2 (lower row) did not react with trypsinized iRBC. The specific antibodies that reacted with the iRBC surface were detected with Alexa Fluor–labeled goat anti-rabbit IgG. Visualization of STEVOR surface antigens. Double staining of live 5A-iRBCs with either anti-S1 (or anti-S2) (panel i) with anti-Glycophorin C antibodies.Niang M, Yan Yam X, Preiser PR. The Plasmodium falciparum STEVOR Multigene Family Mediates Antigenic Variation of the Infected Erythrocyte. PLoS Pathog. 2009;5(2):e1000307. Epub 2009 Feb 20.

STEVOR-specific immunofluorescence staining of mature (>24 h after red blood cell invasion) blood-stage P. falciparum D10 (A and C) and representative field isolates K1657 and K1640 from patients in Kilifi, Kenya (B and D), respectively. Parasites were stained using anti-STEVOR peptide 1 antibodies (A and B) or anti-S1 serum (C and D). Early trophozoite (ET), late trophozoite (LT), and schizont (S) stage parasites are shown. All samples were stained with either rabbit anti-STEVOR peptide 1 affinity-purified antibodies (Stevor) or with anti-S1 rabbit serum (STEVOR), as well as the Maurer’s cleft specific anti-PfSBP1 mouse serum (PfSBP1) and the DNA-specific nuclear stain DAPI (at 1 mg/ml). The individual stains and the merged image (Merge) are shown. STEVOR antibody gave a punctate staining pattern throughout the iRBC, present from the late trophozoite stage and throughout early/mid-stage schizont development in 3D7, D10, and K1657 However, in the highly developed segmented schizont, the staining pattern of STEVOR was more diffuse both around the parasite nuclei and throughout the iRBC cytosol, with some potentially associated with the iRBC surface membrane. Blythe JE, Yam XY, Kuss C, Bozdech Z, Holder AA, Marsh K, Langhorne J, Preiser PR. Plasmodium falciparum STEVOR proteins are highly expressed in patient isolates and located in the surface membranes of infected red blood cells and the apical tips of merozoites. Infect Immun. 2008 76(7):3329-36.

Representative images from fixed and live IFA of iRBCs from different clones. A4(tr-II) clone contains GFP tagged STEVOR. For fixed IFA, cells from 5A, A4 and A4 (tr-I ) clones were first treated with 4% paraformaldehyde for 15 minutes followed by permeabilization wtih 0.05% Triton X100. Permeabilized cells were then incubated with anti-S1 serum as the primary antibody. For 5A, the primary antibody was detected with PE conjugated goat anti-rabbit serum; for A4 and A4(tr-I), Alexa-488 conjugated donkey anti-rabbit serum was used as the secondary antibody. For live IFA, A4 and A4(tr-I) cells, after a 30 minutes incubation with 1% BSA, were directly incubated with anti-S1 serum followed by staining with Alexa-488 conjugated goat anti rabbit secondary antibody. A4(tr-II) cells were not incubated with any STEVOR antibody in live IFA assay. In each case, parasite was stained with 6-diaminido-2-phenylindole (DAPI, 2 mg/mL in PBS). Successive columns from left to right show the bright field, DAPI, STEVOR staining/GFP tag and merged images respectively. A4 clone does not exhibit any staining with anti-S1 serum in fixed and live IFA assays showing the lack of STEVOR proteins. Cells shown are representative of different asexual stages from cell population samples (White scale bar, 5μm; black scale bar, 5μm).Singh H, Madnani K, Lim YB, Cao J, Preiser PR, Lim CT. Expression dynamics and physiologically relevant functional study of STEVOR in asexual stages of Plasmodium falciparum infection. Cell Microbiol. 2017 Jun;19(6)