For intracellular localization, parasites were inoculated into 6-well plate having coverslips confluent with HFF cells. Following overnight incubation, parasites were fixed with 100% methanol. For extracellular localization, freshly lysed parasites were filtered, pelleted, and resuspended in PBS. Thereafter, parasites were added to poly-L-lysine coated cover-slips and allowed to incubate for 30 min at 4°C prior to fixation with 100% methanol. 1% BSA fraction V in PBS was used as blocking agent.
The following primary antisera were used: α-Myc MAb 9E10 (1:50) (Santa-Cruz Biotech), mouse α-Ty (1:500; kindly provided by Chris de Graffenried, Brown University), rabbit α-IMC3(1-120) [1:2,000 (Anderson-White et al., 2011 (link))], mouse α-GFP (Abgent; 1:500). Guinea pig α-AAP4 [1:200 (Engelberg et al., 2020 (link))]. Alexa 488 (A488) or A594 conjugated goat α-mouse, α-rabbit, or α-guinea pig were used as secondary antibodies (1:500) (Invitrogen). DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI). A Zeiss Axiovert 200 M wide-field fluorescence microscope equipped with a α-Plan-Fluar 100x/1.3 NA and 100x/1.45 NA oil objectives and a Hamamatsu C4742-95 CCD camera was used to collect images, which were deconvolved and adjusted for phase contrast using Volocity software (Perkin Elmer, USA).
The following primary antisera were used: α-Myc MAb 9E10 (1:50) (Santa-Cruz Biotech), mouse α-Ty (1:500; kindly provided by Chris de Graffenried, Brown University), rabbit α-IMC3(1-120) [1:2,000 (Anderson-White et al., 2011 (link))], mouse α-GFP (Abgent; 1:500). Guinea pig α-AAP4 [1:200 (Engelberg et al., 2020 (link))]. Alexa 488 (A488) or A594 conjugated goat α-mouse, α-rabbit, or α-guinea pig were used as secondary antibodies (1:500) (Invitrogen). DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI). A Zeiss Axiovert 200 M wide-field fluorescence microscope equipped with a α-Plan-Fluar 100x/1.3 NA and 100x/1.45 NA oil objectives and a Hamamatsu C4742-95 CCD camera was used to collect images, which were deconvolved and adjusted for phase contrast using Volocity software (Perkin Elmer, USA).
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