Alexa fluor 568 antibody

For immunostaining of NF-κB, HeLa-TLR4 dual reporter cells were incubated in blocking solution consisting of 5% bovine serum albumin (BSA, Cell Signaling Technology, Cambridge, UK) and 0.3% Triton X-100 (Merck, Darmstadt, Germany) in PBS for 1 h at RT. Next, cells were incubated with primary anti-NF-κB p65 antibody (D14E12, Cell Signaling Technology) overnight at 4°C. After washing, secondary anti-rabbit Alexa Fluor 568 antibody was applied (Thermo Fisher Scientific) for 1 h at RT. Both antibodies were diluted 1:400 in PBS containing 1% BSA and 0.3% Triton X-100. Cell nuclei were counterstained using 4’,6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific).
Stained cells were imaged using the Opera Phenix High-Content Screening System (PerkinElmer, Waltham, USA) with laser lines for Alexa Fluor 568 at 568 nm excitation and 570 nm—630 nm emission and laser lines for DAPI at 405 nm excitation and 435 nm—480 nm emission. Image processing was done using Harmony software (PerkinElmer). Nuclear masks (DAPI stained cell nuclei) and surrounding ring-like masks (cytoplasm) were identified and the mean fluorescence intensity ratio of nuclear to cytoplasmic NF-κB was determined.

NPCs markers SOX2 and Nestin were detected by imaged-based immunocytochemistry analyses. Cells were plated onto Matrigel-coated (Corning) 12-well plates on cover slips at a density of 0.4 × 10⁶ cells per well. After 2 days, cells were fixed using 4% paraformaldehyde, permeabilized with 0.1% Triton-X in phosphate-buffered saline (PBS), and blocked with 2.5% normal donkey serum (Abcam) in PBS/Tween 20. SOX2 protein was detected using anti-SOX2 antibody (ProteinTech Group) diluted to 1:500, and Nestin protein was detected using anti-Nestin antibody (Sigma-Aldrich) diluted to 1:200, following 1-h incubation at room temperature. Cells were incubated with Alexa Fluor 488 antibody (Thermo Fisher Scientific) diluted to 1:2,000 for Nestin detection and with Alexa Fluor 568 antibody (Thermo Fisher Scientific) diluted to 1:1,000 for SOX2 detection at room temperature for 45 min. Prepared coverslips were mounted onto slides using ProLong™ Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) and imaged using a Nikon A1R confocal microscope (UAB High Resolution Imaging Facility).

To reduce the amount of EdU uptake by replicating host cells, HCT-8 cells from confluent cultures were irradiated at 6,000 rad and stored in liquid nitrogen until further use. Thawed irradiated HCT-8 cells were plated on round coverslips in 24-well culture plates and incubated for ∼24 h. HCT-8 monolayers were infected with excysted sporozoites and washed two times with sterile DPBS at 2 hpi to remove extracellular parasites. Starting at 6 hpi, EdU was added to the culture medium at a final concentration of 10 μM for separate 2-h pulses spaced over 48 h before fixing the cells in 4% formaldehyde for 10 min. Coverslips were permeabilized in 0.05% saponin and treated with the Click-iT Plus EdU 488 imaging kit (Thermo Fisher Scientific) to label EdU. Coverslips were then labeled with anti-RH (a polyclonal antibody generated against Toxoplasma gondii that recognizes all intracellular stages of C. parvum [24 (link)]), followed by labeling with Alexa Fluor 568 antibody (Thermo Fisher Scientific) and Hoechst staining. To calculate the percentage of parasites in each life stage per time point, the number of parasites at each life stage was counted from 10 fields using a 100× oil immersion objective on a Zeiss Axioskop Mot Plus fluorescence microscope, and then the sum was divided by the total number of C. parvum parasites for that time point.