Goat α rat alexafluor 488

Cells were plated and grown in 12-well plates overnight to log phase. Cells were pulsed with 25 μM BrdU for 20 min at 37 °C. The cells were washed with PBS × 2 and treated with media containing 5 mM hydroxyurea (Sigma-Aldrich) for 6 h at 37 °C. The HU was removed, the cells were washed with PBS × 2, and fresh media was added to allow recovery for 0, 4, 6, 8, and 12 h. After recovery, the cells were washed with PBS and harvested using 0.25% trypsin. The cells were fixed with 70% ethanol at -20 °C, pelleted at 1000 g, and rehydrated with 2% BSA/0.1% Tween20/PBS. The DNA was denatured with 3 M HCl for 30 min/RT, and the cells were blocked with 2% BSA/0.1% Tween20/PBS for 30 min/RT. BrdU was stained using a rat anti-BrdU antibody (1:200 rat α-BrdU (clone BU1/75; #MCA2060 BioRad)) for 1 h/RT, followed by incubation with 1:500 goat α-rat AlexaFluor-488 (Life Technologies) in the presence of RNAse for 2 h/RT. Equal numbers of cells were stained with 7-AAD (5ug/mL) and analyzed using a Becton Dickinson FACSCalibur flow cytometer and FlowJo software (https://www.flowjo.com/).

Extracellular parasites were collected and purified as previously (Liu et al., 2014 (link)). Parasites were washed once with buffer A with glucose (BAG, 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO₄, 5.5 mM glucose, and 50 mM HEPES, pH 7.4), and an aliquot of 2 × 10⁴ parasites was overlaid on a coverslip previously treated with poly-L-Lysine. Intracellular tachyzoites were grown on hTERT cells on coverslips. Both extracellular and intracellular parasites were fixed with 3% paraformaldehyde for 20 min at room temperature (RT), permeabilized with 0.3% Triton X-100, blocked with 3% bovine serum albumin (BSA), and exposed to primary antibodies (Ratα-HA 1:100). The secondary antibodies used were goat-αrat Alexa Fluor 488 (Life Technologies) at a 1:1000 dilution. For co-localization studies, we used α-Sag1 (1:1000) as membrane marker and α-TgSERCA as ER marker (1:1000). Slides were examined using an Olympus IX-71 inverted fluorescence microscope with a photometric CoolSNAP HQ charge-coupled device (CCD) camera driven by DeltaVision software (Applied Precision, Seattle, WA). Super-resolution images were imaged with a 63× oil (NA 1.4) objective on an 880-laser scanning microscope with Airyscan (Zeiss, Germany) with a 2× zoom. Airyscan images were process with the Zen Black Software (Zeiss, Germany).