Indirect immunofluorescence studies with rabbit anti-MSP-4 antibodies. P. falciparum-infected erythrocytes were stained with antisera from rabbits immunized with an N-terminal fragment of MSP-4 (from strain w2mef) expressed as a fusion with GST (VM912C). Staining of schizonts shows a typical bunch-of-grapes appearance, which is characteristic of merozoite surface proteins.Marshall VM, Silva A, Foley M, Cranmer S, Wang L, McColl DJ, Kemp DJ, Coppel RL. A second merozoite surface protein (MSP-4) of Plasmodium falciparum that contains an epidermal growth factor-like domain. Infect Immun. 1997 65:4460-7.

Co-localization of PfMAg-1 with PfMSP-4 or PfMSP-119 at late schizont/merozoite parasites examined by confocal fluorescent microscopy. PfMSP-119 is the 19-kDa C-terminal fragment of PfMSP-1 that remains membrane-bound on the invading merozoite Top: Merozoite detected with antibodies X62c (against PfMAg-1) and α-PfMSP-4. Middle: Mature schizont detected with antibodies X62c and α-PfMSP-119. Bottom: Merozoite detected with antibodies X62c and α-PfMSP-119. Bright field images are shown on the left panel. PfMSP-1 and PfMSP-4 are two of the major merozoite surface proteins characterized. Schizonts/merozoite of 3D7 strain, PfMAg-1 co-localized very well with PfMSP-119, and partially with PfMSP-4, in the typical bunch-of-grapes fluorescence pattern haracteristic of merozoite surface proteins.Gao YH, Li HL, Lu Y, Gao FM, Lin YH, Zhou HC, Zhang LH, Wang H. Identification of a vaccine candidate antigen, PfMAg-1, from Plasmodium falciparum with monoclonal antibody M26-32. Parasitol Res. 2009 105:1723-32. PMID: 19777263

Confocal images of FITC PfMAg-1 in mature stage P. falciparum 3D7 infected erythrocytes examined by indirect immunofluorescent assay. Trophozoite stage parasites were immunostained with McAb X62c followed by FITC-labeledsecondary antibody. In addition to the fluorescent labeling on the bodies of trophozoite parasites, dispersed fluorescence patches were observed underneath the parasite-infected erythrocyte membrane (a), while no fluorescence was observed when normal mouse serum was used (b)Co-localization of PfMAg-1 with PfMSP-4 or PfMSP-119 at late schizont/merozoite parasites examined by confocal fluorescent microscopy. a Merozoite detected with antibodies X62c and α-PfMSP-4. b Mature schizont detected with antibodies X62c and α-PfMSP-119. c Merozoite detected with antibodies X62c and α-PfMSP-119. Bright field images are shown on the left panel.Gao YH, Li HL, Lu Y, Gao FM, Lin YH, Zhou HC, Zhang LH, Wang H. Identification of a vaccine candidate antigen, PfMAg-1, from Plasmodium falciparum with monoclonal antibody M26-32. Parasitol Res. 2009 105(6):1723-32.

MSP4 is carried into RBCs during invasion without cleavage. (A) Invading 3D7 merozoites were labeled with PfRON4 (green) and antibodies raised against full-length MSP4 (red). MSP4 labeling was visible on both sides of the tight junction for early-, mid-, and late-stage invading parasites. (B) D10 strain merozoites were labeled with PfRON4 (green) and antibodies raised against different regions of MSP4 (red). All MSP4 antibodies labeled merozoites on both sides of the tight junction. Images are representative of mid- and late-stage invading parasites. N-terminal (anti-MSP4A) and C-terminal (anti-MSP4D) regions of MSP4 were carried into RBCs during invasion, without any detectable cleavage and shedding (C) MSP4 is present at 5 h postinvasion. Isolated 3D7 strain merozoites were allowed to invade and fixed at 10 min and 2 and 5 h postinvasion. Ring-stage parasites were labeled with MSP1-19 antibodies (red) and full-length MSP4 (green). Positive MSP4 labeling was seen at 5 h postinvasion. Antibody concentrations used were 1:500 for secondary antibodies and 1:50 for all MSP4 antibodies.Boyle MJ, Langer C, Chan JA, Hodder AN, Coppel RL, Anders RF, Beeson JG. Sequential processing of merozoite surface proteins during and after erythrocyte invasion by Plasmodium falciparum. Infect Immun. 2014 82(3):924-36.