Paraformaldehyde 0.05 glutaraldehyde

For immunolocalization at the ultrastructural level, infected RBCs were fixed in 4% paraformaldehyde/0.05% glutaraldehyde (Polysciences Inc.) in 100mM PIPES/0.5mM MgCl2, pH 7.2 for 1 hour at 4°C. Samples were then embedded in 10% gelatin and infiltrated overnight with 2.3M 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.). 50 nm sections were blocked with 5% fetal bovine serum/5% normal goat serum for 30 min and subsequently incubated with primary antibodies followed by secondary anti-rabbit conjugated to 18 nm colloidal gold (Jackson ImmunoResearch Laboratories, Inc.). Sections were washed in PIPES buffer followed by a water rinse, and stained with 0.3% uranyl acetate/2% methyl cellulose. Samples were viewed with a JEOL 1200EX transmission electron microscope (JEOL USA Inc.) equipped with an AMT 8 megapixel digital camera (Advanced Microscopy Techniques). All labeling experiments were conducted in parallel with controls omitting the primary antibody. These controls were consistently negative at the concentration of colloidal gold conjugated secondary antibodies used in these studies.