“…Briefly, for IFAs of intracellular parasites, infected confluent HFF monolayers were fixed for 20 min in 4% paraformaldehyde in phosphate‐buffered saline (PBS) or with cold methanol for 6 min, permeabilised with 0.2% Triton X‐100, blocked with 10% fetal calf serum in PBS, and then incubated with primary antibodies: anti‐HA (Roche) 1:100, anti‐GFP (Abcam) 1:2,000, anti‐Myc 1:100 (Santa Cruz Biotechnology), anti‐SAG1 1:1,000 (Couvreur, Sadak, Fortier, & Dubremetz, ), anti‐MIC3 1:500 (Achbarou, Mercereau‐Puijalon, Autheman, et al, ), anti‐proMIC3 1:200 (Cerede, Dubremetz, Bout, & Lebrun, ), anti‐ROP1 1:1,000 (Leriche & Dubremetz, ), anti‐ROP7 1:1,000 (kindly provided by Dr Peter Bradley), anti‐AMA1 1:1,000 (Lamarque et al, ), anti‐GRA3 1:500 (Achbarou, Mercereau‐Puijalon, Sadak, et al, ), anti‐CPL 1:500 (Larson et al, ), anti‐proM2AP 1:400 (kindly provided by Dr Vern Carruthers), anti‐proROP4 1:100 (kindly provided by Dr Gary Ward), anti‐MIC2 1:400 (Achbarou, Mercereau‐Puijalon, Autheman, et al, ), anti‐RON2 1:500 (Besteiro, Michelin, Poncet, Dubremetz, & Lebrun, ), anti‐GAP45 1:2,000 (Frenal et al, ), anti‐GAMA 1:500 (Huynh & Carruthers, ), anti‐MIC5 1:500 (Brydges et al, ), anti‐MIC6 1:1,000 (Reiss et al, ), anti‐MIC10 1:500 (Hoane et al, ), anti‐PLP1 1:500 (Kafsack et al, ), anti‐MIC1 1:500 (Reiss et al, ), anti‐MIC4 1:1,000 (Reiss et al, ), anti‐SUB1 1:500 (Miller, Binder, Blackman, Carruthers, & Kim, ), and anti‐ARO 1:1,000 (Mueller et al, ), followed by goat anti‐rabbit or goat anti‐mouse or goat anti‐rat immunoglobulin G conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes, Invitrogen). Coverslips were mounted onto microscope slides using Immumount (Calbiochem).…”