Immunofluorescence Imaging of Intracellular Parasites

Immunofluorescence assays were performed as previously described^{51 (link)}. Parasite strains of choice were inoculated in six-well plates containing coverslips with HFF cells and fixed with 100% methanol and blocked in 1% BSA in PBS. Alternatively, extracellular parasites were adhered on coverslips coated with poly-Lysine for 1 hr at 37°C. The following primary antibodies were used: rat α-IMC3 1:2000^{49 (link)}, mouse MAb 45:36 α-IMC1 1:1000 (kindly provided by Gary Ward, University of Vermont), α-Myc conjugated with A-488 (Cell Signaling) and rabbit α-human centrin 1:1000 (kindly provided by Iain Cheeseman, Whitehead Institute). Alexa fluorophores A488 and A594 (Invitrogen) conjugated to α-rat, α-rabbit, and α-mouse secondary antibodies were used. Nuclear material was co-stained with 4′6-diamidino-2-phenylindole (DAPI). A Zeiss Axiovert 200 M wide-field fluorescence microscope equipped with a α-Plan-Fluar 100×/1.45 NA oil objective and a Hamamatsu C4742-95 CCD camera was used to collect images, which were deconvolved and adjusted for phase-contrast using Volocity software (Improvision/Perkin Elmer).