Rabbit α ha

To localise expressed proteins incorporating the HA tag, immunofluorescence assays were performed using methodology set out in Tonkin et al. [59 (link)], with minor modifications. When indicated, cells were incubated with 20 nM MitoTracker CMXRos (Sigma) for 30 min at 37°C prior to fixation. Cells were washed once in DPBS before fixing in a solution 4% paraformaldehyde, 0.0075% glutaraldehyde in DPBS. After one wash in DPBS, they were permeabilised in 0.1% Triton X-100/DPBS. Another wash in DPBS was performed, and cells were blocked in 3% BSA/DPBS overnight at 4°C. Primary antibodies diluted in 3% BSA/DPBS were added and incubated for 1 h. Dilutions and antibodies used were as follows: rat α-HA 3F10 (Roche, 1:500), rabbit α-HA (Abcam, 1:500), rabbit α-PfERD2 (MR4, 1:500), rat α-PfAMA1 (MR4, 1:200), rat α-PfBIP (MR4, 1:200), rabbit α-LDH (1:2000). After antibody incubation, cells were washed 3 times in DPBS for 10 min; and the secondary antibodies were added diluted 1:5000 in 3% BSA/DPBS and incubated for 1h. Cells were then washed 3 times in DPBS for 10 min and mounted with ProLong Gold Antifade Mountant with DAPI (ThermoFisher). The slides were sealed and imaged on a DMi8 (Leica Microsystem) using LasX software. Images were processed using ImageJ 1.51k software [60 ]. Our protocol is available on protocols.io [61 ].