Colocalization immunofluorescence of PfMC-2TM with Pf130. Trophozoite and schizont-infected erythrocytes were fixed with ice-cold acetone and incubated sequentially with the primary antibodies SP1C1 and SP1A6 specific for PfMC-2TM and Pf130, respectively. The monoclonal antibodies were contained in spent hybridoma culture supernatant and were used undiluted. The anti-mouse secondary antibodies conjugated to Alexa 488 and Alexa 568 (Molecular Probes) were diluted 1: 1000 in 1× PBS and added separately. Following incubation for 1 h at 37°C, slides were washed three times in 1× PBS and once in distilled water. The smears were mounted in Vectashield (Vector) containing 4′, 6-diamidino-2-phenylindole (DAPI) to stain parasite DNA. Parasites were examined by a Nikon epifluorescence microscope. (A) P. falciparum-infected erythrocytes incubated with mAb SP1A6 followed by anti-mouse 488 Alexa conjugate (green). (B) P. falciparum schizont-infected erythrocytes incubated with mAb SP1C1, followed by anti-mouse 568 Alexa conjugate (red). (C) Parasite nuclei counterstained by DAPI. (D) Overlay of SP1A6 and SP1C1 antibodies showing colocalization (yellow) of PfMC-2TM and Pf130 Maurer's cleft proteins.
