Ultracut uct cryo ultramicrotome

For IEM, mature schizonts from the DPAP3-GFP and 3D7 control lines were concentrated using a magnetic activated cell sorting (MACS) LD separation column (Miltenyi Biotec). Briefly, iRBCs were loaded onto an LD column attached to Midi MACS pre-equilibrated with media. The column was washed twice with media and schizonts eluted with media after detaching the column from the magnet. Parasites were then fixed in 4% paraformaldehyde/0.1% glutaraldehyde (Polysciences) in 100 mM PIPES and 0.5 mM MgCl₂, pH 7.2, for 1 h at 4°C. Samples were embedded in 10% gelatine and infiltrated overnight with 2.3 M sucrose/20% polyvinyl pyrrolidone in PIPES/MgCl₂ at 4°C. Samples were trimmed, frozen in liquid nitrogen, and sectioned with a Leica Ultracut UCT cryo-ultramicrotome (Leica Microsystems). Sections of 70 nm were blocked with 5% FBS and 5% NGS for 30 min and subsequently incubated with rabbit anti-GFP antibody 6556 (Abcam) at 1:750 overnight at 4°C. Colloidal gold conjugated anti rabbit (12 nm) IgG (Jack Imm Res Lab) was used as secondary antibody.

Samples for immunoEM were prepared as before^{3 (link)}. Briefly, the infected RBCs were fixed in 4% paraformaldehyde (Polysciences Inc., Warrington, PA) with 100 mM PIPES/0.5 mM MgCl₂, pH 7.2 for 1 h at 4 °C. Samples were then embedded in 10% gelatin and infiltrated overnight with 2.3 M sucrose/20% polyvinyl pyrrolidone in PIPES/MgCl2 at 4 °C. Samples were trimmed, frozen in liquid nitrogen, and sectioned with a Leica Ultracut UCT cryo ultramicrotome (Leica Microsystems Inc., Bannockburn, IL). 50 nm sections were blocked with 5% FBS/5% NGS for 30 min and subsequently incubated with primary antibodies for 1 h, followed by secondary antibodies conjugated to 12 nm or 18 nm colloidal gold (1:30) for 1 h. Sections were washed in PIPES buffer followed by a water rinse, and stained with 0.3% uranyl acetate/2% methyl cellulose and viewed on a JEOL 1200EX transmission electron microscope (JEOL USA, Peabody, MA) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA). All labeling experiments were conducted in parallel with controls omitting the primary antibody, which were consistently negative at the concentration of colloidal gold-conjugated secondary antibodies used in these studies.