Immunofluorescence Assay (IFA)

Immunofluorescence assays were performed as described earlier²⁵. Parasites were washed twice with PBS and fixed using 4% paraformaldehyde (ProSciTech, Electron Microscopy-EM grade) and 0.0075% glutaraldehyde (EM grade) in PBS at room temperature for 30 min. Fixed cells were washed 2 times with PBS and permeabilized using 0.1% Triton X-100 at room temperature for 3 min. Cells were washed again twice with PBS followed by blocking with 3%BSA at 4 °C for 1 h. The primary antibodies were used at the following dilutions; rabbit anti-PfAtg8 (1:600), rabbit anti-PfAtg5 (1:400), mouse anti-SSB protein (1:400) and mouse anti-KDEL (1:400) in 3% BSA in PBS for 1 h at room temperature. Secondary antibodies used were Alexa Fluor 488- and 568-conjugated goat anti-rabbit and goat anti-mouse (Molecular Probes) respectively at 1:200 dilutions in 3% BSA for 1 h at room temperature. Nucleus was stained with Hoechst 33258 (Sigma). Parasites were mounted over the glass slide using ProLong Gold antifade mountant (Molecular Probes). Images were acquired with Carl Zeiss LSM700 or DeltaVision (Applied Precision, GE) microscope. Software used were Zen and SoftWoRx respectively using a 100X oil immersion phase contrast objective. DIC images were taken with polarized light.