Nol lsm 710

The immunofluorescence staining was performed as previously described (55 (link)). Briefly, the EPC monolayers were infected with WT eseQ-2HA::kan strain, WT trx2-2HA::kan strain, and WT eseJ-2HA::kan strain at MOI 10. At 5 hpi, the EPC monolayers were fixed in 4% PFA and stained with rabbit anti-HA (Cell Signaling Technology) at 1:200, mouse anti-LPS at 1:200 (56 (link)), goat anti-rabbit IgG (Alexa 488; Molecular Probes) at 1:200, and goat anti-mouse IgG (Alexa 594; Molecular Probes) at 1:200 and sealed with anti-fading mounting reagent with DAPI before images were photographed under a confocal microscope (NOL-LSM 710; Carl Zeiss).
To determine the nucleus accumulation of p65, J774A.1 cells were infected with WT/RFP, Δtrx2/RFP, and Δtrx2[trx2]/RFP strains at MOI 10 for 2 hours. After fixation, the monolayers were stained with mouse anti-p65 (Cell Signaling Technology) at 1:200, and goat anti-mouse IgG (Alexa 594; Molecular Probes) at 1:200 and sealed with anti-fading mounting reagent with DAPI before images were photographed under a confocal microscope (NOL-LSM 710; Carl Zeiss).

Seed the cells in 35 mm plates (density: 1 × 10⁴) for incubation overnight. Replace 1 mL serum-free medium containing Cy5 labeled mAb_Nectin-4 (20 µg/mL) to each plate and incubation was performed for 1 h. Rinse the Cy5-treated cells with pre-cooling PBS for three times, 200 μL 4% paraformaldehyde solution was added to fix cells. Then DAPI and FITC-Phalloidin was used for nuclear and cytoskeleton staining respectively. Images with different channels (Excitation/Emission of the Cy5, DAPI, and FITC were 650/670 nm, 360/450 nm, and 490/520 nm, respectively) were acquired by fluorescence optics of a confocal microscope (Zeiss, NOL-LSM 710). For blocking studies, MDA-MB-468 cells were incubated with excess mAb_Nectin-4 for 1 h previously before experiments.

The ovulated eggs of the maternal individual from strain F were inseminated by sperm of a genotypic male from strain A⁺, a temperature-dependent male from strain A⁺, and a male common carp, respectively. The fertilized eggs were digested by 0.25% trypsin to remove their shells and then incubated at 24°C. The fertilized eggs at different developmental stages were fixed with 4% paraformaldehyde in PBS at 4°C overnight on a working shaker. About 40 inseminated eggs were fixed at each time point as previously described (Zhang et al., 2015 (link)). After washing with PBS three times, the nuclei were stained using DAPI (Sigma, United States), and the images were obtained via confocal microscopy (NOL-LSM 710 Carl Zeiss) as described (Zhu et al., 2018 (link)).