Ab138698

HFFs were grown to confluence in an 8-well μ-slide (Ibidi) and infected with T. gondii strains for 24 h. The slides were fixed with 4% w/v formaldehyde (Sigma) in phosphate-buffered saline (PBS) (Sigma). The cells were permeabilised with 0.2% v/v Triton X-100 (Sigma) for 15 min or 1 min and blocked with 2% w/v bovine serum albumin (Sigma) for 1 h. The cells were stained with 1:500 rat anti-HA (Roche #11867423001), followed by 1:1000 goat anti-rat 594 (Invitrogen #A11007), followed by a mixture of 1:500 mouse anti-ROP1 (Abnova #MAB17504) and 1:1000 rabbit anti-T. gondii (Abcam #ab138698), and finally with a mixture of 1:1000 goat anti-mouse 488 (Invitrogen #A11029), 1:1000 goat anti-rabbit 647 (Invitrogen #A21244), and 5 μg/mL DAPI (Sigma), each for 1h at room temperature. Images were acquired on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon CFI APO TIRF 100x/1.49 objective and Hamamatsu C11440 ORCA Flash 4.0 camera running NIS Elements (Nikon).

150,000 BMDMs per well were seeded in an 8-well μ-slide and stimulated with 10 ng/mL (~100 U/mL) recombinant mouse IFNγ for 24 h prior to infection. The BMDMs were infected with parasite strains at an MOI of 0.3 for 3 h, then washed and fixed with 4% w/v formaldehyde for 15 min. Prior to permeabilisation, the cells were blocked with 2% w/v BSA for 1 h and extracellular parasites were stained with 1:1000 rabbit anti-T. gondii (Abcam #ab138698) for 1 h at room temperature followed by 1:1000 goat anti-rabbit 405 (Invitrogen #A31556) for 1 h at room temperature. The cells were then permeabilsed with 0.2% v/v Triton X-100 for 15 minutes, blocked again with 2% w/v BSA for 1 h, stained with 1:200 mouse anti-ubiquitinylated proteins (Sigma #04–263) overnight at 4°C, and finally stained with 1:1000 goat anti-mouse 488 (Invitrogen #A11029) for 1 h at room temperature. Nine tiled fields of view were captured for each well on a Nikon Ti-E inverted widefield fluorescence microscope as above. The images were blinded, and the percentage of ubiquitinated vacuoles was determined manually using ImageJ, excluding T. gondii cells which were positive for extracellular staining. A median of 290 vacuoles were analysed per strain per replicate. Differences between strains were determined by two-sided t-test with Benjamini-Hochberg adjustment.