Eclipse c1 plus

Cells were seeded on glass coverslips in a 12–well plate and transfected with the indicated plasmid for 24 h. Then, the cells were treated with or without DNA damage reagents according to the experimental setup. The cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min at RT and then permeabilized in 0.5% Triton X–100 for 10 min. Slides were blocked in 5% normal goat serum (NGS) and incubated with primary antibodies diluted in 1% NGS overnight at 4 °C. Samples were then incubated with the indicated secondary antibodies labeled with Alexa Fluor 488 or 555 (Invitrogen) diluted in 1% NGS at RT for 1 h. Thereafter, they were stained with DAPI for 15 min at RT. Coverslips were mounted using Dako Fluorescence Mounting Medium (Agilent) and imaged using a Nikon confocal microscope (Eclipse C1 Plus). All scoring was performed under blinded conditions.

Control and Met-treated protoscoleces in different times (6-12-24-36 and 48 h) were incubated with 10 mg/mL JC-1 dye for 30 min at room temperature. After incubation, parasites were washed with 20 mM HEPES buffer, pH 7.2, and images were taken using a confocal microscope (Nikon Eclipse C1 Plus). The intensities of green (excitation/emission wavelength = 485/538 nm) and red (excitation/emission wavelength = 485/590 nm) fluorescence were analyzed for 20 individual protoscoleces from control and treated-samples. Images were analyzed using Image J software (NIH). The ratio of red to green fluorescence of JC-1 images was calculated using NIH Image J software (http://rsb.info.nih.gov/ij/).

Cells were seeded and cultured on glass coverslips in 12‐well plate and fixed in 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature. Cells were permeabilized in 0.5% Triton X‐100 for 10 min. Slides were blocked in 5% normal goat serum (NGS) and incubated with primary antibodies diluted in 1% NGS overnight at 4°C. Samples were then incubated with secondary antibodies labeled with Alexa Fluor 488 (Invitrogen) diluted in 1% NGS at RT for 1 h.
Thereafter, they were stained with DAPI (or plus Phalloidin) for 15 min at room temperature. Coverslips were mounted using Dako Fluorescence Mounting Medium (Agilent) and imaged using Nikon confocal (Eclipse C1 Plus). All scoring was performed under blinded conditions.
For quantification of the nuclear cGAS percentage, we used ImageJ software to quantify the nuclear cGAS immunofluorescence intensity relative to whole‐cell cGAS intensity from 6 different fields with n > 50 cells. Percentage of nuclear cGAS = (nuclear cGAS/whole‐cell cGAS intensity) X100%.

We fixed a small piece of the insert membrane with 3.7% paraformaldehyde in PBS, and permeabilized with 0.2% Triton X-100 in PBS, followed by direct staining with an anti-HBoV1 NS1C antibody [23 (link)]. Immunofluorescence analysis was performed using a method described previously [17 (link)]. Confocal images were taken with an Eclipse C1 Plus confocal microscope (Nikon, Tokyo, Japan) controlled by Nikon EZ-C1 software. DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus.

Cells were seeded and cultured on glass coverslips in 12‐well plate and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. Cells were permeabilized in 0.5% Triton X‐100 for 10 min. Slides were blocked in 5% normal goat serum (NGS) and incubated with primary antibodies diluted in 1% NGS overnight at 4 °C. Samples were then incubated with indicated secondary antibodies diluted in 1% NGS at RT for 1 h, before staining with DAPI for 15 min at room temperature. Coverslips were mounted using Dako Fluorescence Mounting Medium (Agilent) and imaged using Nikon confocal microscope (Eclipse C1 Plus). All scoring was performed under blinded conditions. γ‐H2AX, BRCA1, and 53BP1 foci were counted from 30 microscopic fields containing approx. 300 cells from 3 independent experiments per time point.