Tachyzoites treated with IAA or vehicle for three growth cycles were inoculated onto coverslips with confluent monolayers of HFFs and grown for 24 h. Cells were fixed in 4% (vol/vol) paraformaldehyde/PBS (Sigma-Aldrich) for 10 min and permeabilised in 0.1% (vol/vol) Triton X-100/PBS before blocking in 3% (wt/vol) BSA/PBS (Sigma-Aldrich) for 1 h. Cells were incubated with primary antibodies used at 1:1,000 in 3% BSA/PBS for 1 h. Primary antibodies used were as follows: rat α-HA 3F10 (1:1,000, Cat# 11867423001, RRID:AB_390918; Roche); mouse α-SAG1 DG52 (Burg et al, 1988 (link)); rabbit α-GAP45 (1:1,000) (Gaskins et al, 2004 (link)); rabbit α-ACP (Waller et al, 1998 (link)); and rabbit α-IMC1 (Ward & Carey, 1999 (link)) (Table S7). Cells were washed 3X in PBS and probed for 1 h with Alexa Fluor–conjugated secondary antibodies (Thermo Fisher Scientific; see Table S8 for full list), plus 5 μg/ml DAPI used at 1:1,000 in 3% BSA/PBS. Coverslips were washed and mounted onto glass microscope slides with Vectashield (Vector Labs). Parasites were imaged on a Zeiss Live Cell Axio Observer widefield microscope. Images were processed in ImageJ (Schneider et al, 2012 (link)).
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