C4742 95 ccd camera

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).

Conventional TEM was performed as described previously (Ichimura et al., 2009 (link), 2010 (link)). In brief, the slices of fixed kidney cortex were successively immersed in 0.4% osmium tetroxide (OsO₄) in 0.1 M PB for 1 h, 2% low-molecular-weight tannic acid (LMW-TA, Electron Microscopy Sciences, Hatfield, PA, United States) in 0.05 M maleate buffer for 4 h, and 1% uranyl acetate in 0.05 M maleate buffer for 3 h. The slices were then dehydrated with a graded series of ethanol and embedded in Oken Epok 812 epoxy resin (Oken-shoji, Tokyo, Japan). Ultrathin sections (90–100 nm thickness) were produced with an ultra 45° diamond knife (Diatome, Biel, Switzerland) and transferred to 50-mesh copper grids coated with a Formvar membrane. The ultrathin sections stained with lead citrate and uranyl acetate were digitally photographed with an H-7100 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan), which was equipped with a C4742-95 CCD camera (Hamamatsu Photonics, Shizuoka, Japan).