Paraformaldehyde (pfa)

For postnatal pups or adult animals, 185 mg/kg sodium pentobarbitone was injected intraperitoneally. Animals were then transcardially perfused with 0.9% saline solution (0.9% w/v NaCl in MilliQ^TM water; Millipore, Billerica, USA), followed by freshly prepared 4% w/v paraformaldehyde (PFA; ProSciTech, Thuringowa Central, Australia) in phosphate-buffered saline (PBS; pH 7.4; Lonza, Basel, Switzerland). The head was removed and post-fixed at 4 °C in 4% PFA in PBS until required for tissue processing. For tangential sections, animals were transcardially perfused with 0.9% saline solution to remove the blood from the brain. The cortices were then dissected and flattened between two slides approximately 1 mm apart and fixed in 4% PFA in PBS at 4 °C for at least 48 h. Following fixation, the flattened cortices were transferred into PBS and maintained at 4 °C until required for sectioning. Prior to sectioning, brains were blocked in 3% w/v Difco™ Noble agar (Becton, Dickinson and Company, Franklin Lakes, USA) in MilliQ water. Free-floating sections of 50-μm thickness were cut using a vibratome (Leica Biosystems, Jurong, Singapore).

Fluorescence images of parasites were captured using a GE Applied Precision Deltavision Elite microscope with 100x 1.4NA objective and with a sCMOS camera and deconvolved with the SoftWorx software. Chromatic calibration of the microscope was performed prior to imaging experiments. For immunofluorescence assays, parasites were fixed on slides using 4% paraformaldehyde (ProSciTech) (127 (link)) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). After blocking in 3% bovine serum albumin (Sigma Aldrich) the cells were incubated for 1 h with rabbit polyclonal anti-PfERD2 (1:2000) (128 (link)), mouse monoclonal anti-HA (Cedarlane, CLH104AP, 1:2000), goat anti-human Hb (1:1000, Cedarlane) or rabbit polyclonal anti-BiP (1:1000) (125 (link)). Bound antibodies were then visualized with either Alexa Fluor-594 anti-rabbit, anti-mouse or anti-goat IgG and Alexa Fluor-488 anti-mouse or anti-rabbit IgG diluted 1:1000 (Cedarlane). Parasites were mounted in Vectashield (Vecta Laboratories) containing 0.1 μg/ml 4', 6–diamidino-2-phenylindole (Dapi, Invitrogen). Images shown represent a single optical slice from a deconvolved z-stack. For the LysoTracker labeling experiment, PfPX1-GFP parasites were incubated for 2 h with 75 nM of LysoTracker Red DND-99 (Invitrogen).

For chloroplast ultrastructure, leaf tissue (2 × 2 mm) was cut and fixed in 2.5% (v/v) glutaraldehyde/2% (v/v) paraformaldehyde (ProSciTech, Thuringowa Central, QLD, Australia) overnight, washed three times in 0.1 M phosphate buffer (423 mM NaH₂PO₄, 577 mM Na₂HPO₄, pH 7.2) and then incubated in secondary fixative [1 % (w/v) OsO₄] for 4 h. Leaf tissue was then serially dehydrated and embedded in LR white resin⁷³ (ProSciTech). Approximately 70–80 nm ultrathin resin sections were cut and stained with 2% (w/v) uranyl acetate and lead citrate. For purified carboxysomes, protein samples in TEMB were mounted directly onto TEM grids and negatively stained with 2% (w/v) uranyl acetate. Both leaf sections and purified carboxysomes were observed at 100 kV using a Hitachi HM7100 TEM.

The parasites were fixed using 4% paraformaldehyde (ProSciTech, EM grade) and 0.0075% glutaraldehyde in PBS at room temperature for 30 min. Fixed cells were washed twice with PBS and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Following PBS washes, cells were blocked in PBS containing 3% BSA for 1 h at 4 °C. This protocol was followed for the first primary antibody anti-PfAtg5 (1:200), Alexa Flour 568-conjugated goat anti-rabbit (1:200) was used as secondary antibody. Zenon rabbit IgG labeling was used to label PfAtg8. Zenon complex IgG 488 was prepared as recommended by the supplier (Zenon® Alexa Fluor® 488 Rabbit IgG Labeling Kit, ThermoFisher Scientific) at a molar ratio of 6:1. Zenon complex thus formed was incubated for 1 h followed by fixation by 4% paraformaldehyde and 0.0075% glutaraldehyde in PBS at room temperature for 30 min. Nuclear staining was performed with Hoechst 33258 (1:300) for 10 min. Parasites were immediately mounted on the glass slide using VECTASHIELD (Vector Laboratories) mountant and imaging was carried out using Zeiss LSM 700 confocal microscope.

Fluorescence images of parasites were captured using a GE Applied Precision Deltavision Elite microscope with 100×1.4NA objective and with a sCMOS camera and deconvolved with the SoftWorx software. Pearson’s correlation coefficients were calculated with the Fiji software^{49 (link)} on regions of interests of image stacks, including zero-zero pixels and without thresholding. For the colocalisation analysis between PfSAC1 and the ER marker Bip, Mander’s correlation coefficients were calculated with the Fiji software on regions of interests of image stacks, with manual thresholding. Chromatic calibration of the microscope was performed prior to imaging experiments. For immunofluorescence assays, parasites were fixed on slides using 4% paraformaldehyde (ProSciTech)^{50 (link)}. After blocking in 3% bovine serum albumin (Sigma Aldrich) the cells were incubated for 1 hour with rabbit polyclonal anti-ERD2 1:2000^{34 (link)}, mouse monoclonal anti-HA 1:2000 (Cedarlane, HA.C5), or rabbit polyclonal anti-Bip 1:500 (Sabrina Absalon and Jeffrey Dvorin, unpublished). Bound antibodies were then visualised with Alexa Fluor-594 anti-rabbit IgG and Alexa Fluor-488 anti-mouse IgG diluted 1:1000. Parasites were mounted in Vectashield (Vecta Laboratories) containing with 0.1 μg/ml 4′, 6–diamidino-2-phenylindole (Dapi, Invitrogen). Images shown represent the maximum projection of all z stacks.