Immunocytochemistry and Immunofluorescence Protocols

Immunocytochemistry was performed as described by Kilmartin and Adams (51 (link)). Briefly, yeast were grown to an optical density (OD) equivalent to log phase and were prepared for immunostaining by removing the cell wall. The cell suspension was spotted on polylysine-coated microscope slides, dried, fixed, and permeabilized (10-min incubation in phosphate-buffered saline [PBS] with 0.1% Triton X-100); 1 μg/mL primary antibody (mouse anti-SERCA1a; Abcam) was added and incubated overnight at 4°C, and then 1 μg/mL secondary antibody (Texas Red-tagged goat anti-mouse IgG) was added and incubated for at least 2 h. The slides were stained with 5 μM 3,3′-dihexyloxacarbocyanine [DiOC₆(3)] for 20 min to stain the ER. Antifade mounting medium plus 4′,6-diamidino-2-phenylindole (DAPI) was added before the slides were sealed. Yeast were visualized with a Zeiss confocal LSM 510 microscope.
Immunofluorescence with P. falciparum parasites was performed as described by Pulcini et al. (13 (link)), using the paraformaldehyde/glutaraldehyde fixation method. Parasites were labeled as described by Wang et al. (18 (link)), using 500 nM DHA-biotin probe. Parasites were visualized with a Zeiss confocal LSM 510 microscope.