Dylight 649

Cells were seeded at a density of 2 × 10⁴/well on 4-well Lab-Teck II chamber slides (Nunc, Naperville, IL, USA). After indicated treatment, cells were washed twice with ice-cold PBS and fixed immediately with 4% paraformaldehyde in PBS for 15 minutes at room temperature. After two washes with PBS, cells were permeabilized with 0.25% Triton X-100 for 10 minutes at room temperature. Then cells were blocked with 1% bovine serum albumin (BSA) in PBS/Tween 20 for 30 minutes at room temperature and further incubated overnight with the primary antibodies against TrxR1 (Abcam, 1:300), CD44-PE (eBioscience, 1:160), E-Cadherin (BioLegend, 1:300) or N-Cadherin (Biolegend, 1:300) diluted in 1% BSA in PBS/Tween 20 in a humidified chamber at 4 °C. After three rinses with PBS, secondary antibodies conjugated with Dylight 488, Dylight 649 or Dylight 549 (Abbkine, 1:300) diluted in 1% BSA in PBS/Tween 20 (1:200) was added and incubated for 1 hour at room temperature in dark followed by three washes with PBS. Subsequently, nucleus were counterstained with 4′,6′-diamino-2-phenylindole (DAPI) for 10 minutes. Images were taken with a laser scanning confocal microscope (Nikon A1, Japan) and analyzed by NIS-Elements Viewer 4.20 software.

Healthy flounder (P. olivaceus) of 50 ± 5.8 g or 1 ± 0.1 kg were purchased from a farm in Rizhao, Shandong Province, PR China, and maintained in the aerated recirculating seawater system at 20 ± 2°C for 2 weeks. The flounder with no clinical symptoms determined by general appearance and level of activity were used in the following experiments.
New Zealand white rabbits (∼1 kg) and Balb/C (~30 g) mice were purchased from Qingdao Animal Experimental Center (Shandong, China) and then used for antibody production.
HINAE cells, provided by Dr. Ikuo Hirono of Tokyo University of Marine Science and Technology (37 ), were cultured in Leibovitz’s L-15 medium containing 10% FBS, 100 IU/ml penicillin, and 100 μg/ml streptomycin.
Mouse polyclonal antibodies against flounder IL-2 (diluted at 1:500) and monoclonal antibodies against flounder IgM (diluted at 1:1,000), CD4-1 (diluted at 1:1,000), and CD4-2 (diluted at 1:1,000) were previously produced in our laboratory (38 (link)–40 (link)). The secondary antibodies included DyLight 488 (Abbkine; diluted at 1:1,000) rabbit or mouse IgG, DyLight 649 (Abbkine; diluted at 1:1,000) rabbit or mouse IgG, alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (H + L) (Abbkine; diluted at 1:5,000), and HRP-conjugated goat anti-mouse IgG (Abbkine; diluted at 1:5,000).

The leukocytes were isolated from peripheral blood by 34% (1.050 g/ml) to 51% (1.072 g/ml) v/v discontinuous Percoll density gradient. After being adjusted to 1 × 10⁶ cells/ml, 50 μl of cells was adhered to the slides for 2 h and then fixed with 4% paraformaldehyde for 20 min. The cells were blocked with 5% BSA at 37°C for 1 h. The CD80/86-specific antibodies mixed with mouse polyclonal antibodies MHCII (diluted 1:500), CD40, CD83, and monoclonal antibodies against flounder IgM, CD4-1, and CD4-2 were used as primary Abs at 37°C for 1.5 h. After three washes with PBS-T, the primary Abs were detected by Dylight 488 (Abbkine) rabbit or mouse IgG and Dylight 649 rabbit or mouse IgG. The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI) at room temperature for 20 min. All the Abs were diluted 1:1,000 and unimmunized serum was used as negative controls. Fluorescence images of the samples were obtained using a fluorescence microscope (Olympus DP70, Japan).