Alexa 488 or 546

Cells were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C. After washing with PBS and treatment with 0.2% trypsin in PBS (PBST) for 10 min at 4°C, cells were pre-incubated with blocking buffer (10% goat serum in PBS) for 30 min at room temperature, and then reacted with primary antibodies in blocking buffer for 12 h at 4°C. Followed by washing with 0.2% PBST, cells were incubated with secondary antibodies; anti-rabbit or anti-mouse IgG conjugated with Alexa 488 or 546 (1:300) (Invitrogen) in blocking buffer for 1 h at room temperature. Then, the cells were counterstained with DAPI and mounted.

HepaMN cells were fixed in 4% paraformaldehyde for 10 min at 4 °C. After washing with PBS and treatment with 0.1% Triton-X100 for 10 min at room temperature, cells were pre-incubated with blocking buffer (1% BSA in PBS) for 30 min at room temperature, and then reacted with primary antibodies in blocking buffer for 12 h at 4 °C. Followed by washing with PBS, cells were incubated with secondary antibodies; anti-goat or anti-mouse IgG conjugated with Alexa 488 or 546 (1:1000) (Invitrogen) in blocking buffer for 30 min at room temperature. Then the cells were counterstained with DAPI and mounted.

For immunofluorescence assays, LLC-MK₂ cells infected with tachyzoites at a 10:1 ratio of parasites to host cells were treated with Et-Cipro, Adam-Cipro and Ph-Cipro compounds for 24 h. For Cipro assay, cells were infected in a ratio of 5:1 parasites per host cell and treated for 24, 48 and 72h. After treatment, infected cells were fixed in 3.7% freshly-prepared formaldehyde, permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 3% bovine serum albumin (BSA) in PBS at pH 7.4 and room temperature for 1 h. All antibody incubations were performed in 3% BSA in PBS, for 1h, at room temperature. The following primary antibodies were used: anti-SAG1 (kindly provided by Dr. John Boothroyd, Stanford University School of Medicine, USA), at a dilution of 1:1000; anti-HSP60 and anti-Morn1 (kindly provided by Dr. Boris Striepen, University of Georgia, USA), diluted to 1:2000 and 1:100, respectively; and anti-IMC1 (kindly provided by Dr. Gary Ward, University of Vermont, USA), diluted to 1:1000. The secondary antibodies used here were anti-mouse/rabbit coupled to Alexa 488 or 546 (Molecular Probes). Sytox green (Molecular Probes) and DAPI (Sigma-Aldrich) were used to label the DNA. After labeling, the coverslips were mounted onto slides using Prolong gold (Life Technologies), and samples were examined on a TCSSP5 Leica or Zeiss LSM710 laser scanning confocal microscopes.

All methods for IHC and IF of mouse tumors have been described^{33 (link),62 (link)}. Briefly, tumors were excised at days 16–18 after tumor cell inoculation and snap-frozen in Tissue Freezing Medium (OCT; Jung) or fixed with 10% neutral buffered formalin (3 h) and paraffin embedded. Sections (5–20 μm) were prepared and used directly (frozen tissue) or deparaffinized, rehydrated, and subjected to heat-induced epitope retrieval in citrate buffer (0.01 M sodium citrate pH 6; 20 min). For immunostaining, the following rabbit antibodies were used: anti-CD31 (MEC13.3, BD Bioscience), -SOD3 (Cloud Clone), -VEC (LS-B2138, LSBio), -HIF-2α (PAI-32216, Thermo Fisher Scientific), and -3-NT (#06–284, Cell Signaling). Sections were incubated with appropriate fluorescently conjugated secondary antibodies (Alexa 488 or 546, Molecular Probes) or with peroxidase-labeled anti-rabbit IgG (Dako), followed by amino ethyl carbazol (AEC; Enzo) and hematoxylin counterstaining. Sections were mounted with 4,6-diamidino-2-phenylindole (DAPI)-containing Fluoromount-G (SouthernBiotech; fluorescence analyses) or Dako Faramount Aqueous Mounting Media (Dako; conventional IHC) and analyzed with an Olympus FluoView 1000 confocal microscope with a ×60 1.4 oil plan-Apo objective or with a Leica (DM RB) microscope equipped with an Olympus DP70 camera.

Cultured cells were fixed with 4% formaldehyde for 10 min at 4 °C, subsequently permeabilized in 0.1% triton-X (MP Biomedicals, USA) for 5 min at room temperature and blocked with PBS containing 10% goat serum for 1 h at 37 °C. Incubations with primary antibodies to MyHC (1:200; eBioscience, #14-6503-82), c-casp3 (1:200; Cell Signaling Technologies, Danvers, MA, USA #9664), and MYOD1 (1:200; Santa Cruz, sc-32758) were performed overnight at 4 °C. After washing with PBS, they were incubated with a secondary antibody conjugated with Alexa-488 or -546 (1:300; Molecular Probes) for 30 min at room temperature. Following the manufacturer's instructions, we performed EdU staining with a Click-iT EdU imaging kit (Invitrogen) and stained nuclei with 4,6-diamidino-2-phenylindole (DAPI). We analyzed the stained cells using a BZ-X810 fluorescence microscope (Keyence, Osaka, Japan). We calculated the fusion index as a percentage of nuclei within MyHC-positive myotubes in nine randomly selected images from three healthy individuals.