Deltavision elite imaging microscope

TAB004 conjugation to pHrodo Red was performed using the pHrodo Red, succinimidyl ester (pHrodo Red, SE) kit (P36600, Molecular Probes). TAB004 conjugation to indocyanine green (ICG) was performed using the ICG Labeling Kit –NH₂ (LK31-10, Dojindo Molecular Technologies, Inc.). All conjugations were performed using manufacturer protocols. Cell lines were plated into 4-chamber well slides (154,917, LAB-TEK) at optimal concentration to ensure cells would not be over confluent after 24 h. 24 h after cells were plated, they were treated with 5 μl of TAB004-phRodo Red conjugation solution for various time points at 37 °C, > 90% humidity, and 5% CO₂ conditions. During the last 5 min of treatment, Wheat Germ Agglutinin-Alexa Fluor 488 conjugate (W11261, Molecular Probes), was added to each chamber at 5 μg/ml. The cells were washed with PBS for 5 min (3×) and fixed with 4% formaldehyde. Prolong Gold Antifade reagent with DAPI (P36935, Molecular Probes) was applied to mount coverslips. Images were acquired on a GE Healthcare Life Sciences DeltaVision Elite Imaging microscope.

iRBC infected with parasites expressing the DNMT2-3HA episome were fixed with 0.0075% glutaraldehyde and 4% paraformaldehyde in PBS for 30 min at room temperature. Free aldehydes were quenched with 50 mM NH₄Cl for 10 min at room temperature, and iRBC were permeabilized with 0.1% Triton X-100 for 15 min at room temperature. Permeabilized iRBC were then incubated with primary rat anti-HA antibody (Roche; 3F10) ON at 4°C. After several washes in PBS–0.05% Tween 20, goat anti-rat antibody conjugated to Alexa Fluor 647 was added for 45 min at room temperature. Samples were washed, 5 μl of cells was mounted with Vectashield fluorescence mounting medium, and images were acquired on a Deltavision Elite imaging microscope (GE Healthcare). Images were processed using ImageJ software.