Dylight 488

Immunofluorescence staining was conducted to characterize the expression and distribution of HIF-1α and LOXL2 in DPSCs. DPSCs were seeded on coverslips in 24-well plates (1.5 × 10⁴ cells per well) and cultured at 37 °C overnight. The culture media were replaced with serum-free media and cells were cultured under normoxia or hypoxia for 48 h. Cells were fixed with 4% PFA for 20 min and permeabilized with 0.3% Triton X-100 in PBS for 10 min. After blockading in 10% goat serum for 1 h, cells were incubated overnight with primary antibodies against HIF-1α (1:100; Proteintech, Chicago, IL, USA) and LOXL2 (1:100; Abcam, Cambridge, UK) at 4 °C. The next day, after washing with PBS, cells were incubated with fluorescein-conjugated goat anti-mouse antibody (1:200, DyLight 488; Abbkine, Redlands, CA, USA) and goat anti-rabbit antibody (1:200, DyLight 594; Abbkine, Redlands, CA, USA) at room temperature for 1 h. Finally, nuclei were counterstained with DAPI (Beyotime, Shanghai, China) for 5 min. Images were captured with a confocal microscope (Olympus, Tokyo, Japan) and fluorescent quantitative analysis was performed with ImageJ software.

The cells were washed and fixed using paraformaldehyde at a concentration of 4%, then incubated with the diluted primary antibodies at 4°C overnight. The primary antibodies used are anti-NOS2 and anti-Arg-1 (rabbit polyclonal, 1:300, both from Proteintech, Chicago, IL, United States), p-STAT1 (rabbit polyclonal, 1:200, Affinity, Shanghai, China). After washing of cells with 1×PBS, cells were incubated with goat anti-rabbit IgG Dylight 594 or Dylight 488 (1:500, both from Abbkine, Wuhan, China) for 1 h in the dark. Representative pictures were captured with a Zeiss Imager M2 microscope and were analyzed using Image J software (version: v1.8.0).

A total of 3 × 10⁴ MC3T3-E1 cells or stable virus-transfected strain were seeded on each glass coverslip (Solarbio, China). After 72 h of transfection, cells were fixed with 4% paraformaldehyde before being permeabilized for 20 min with 0.2% Triton-X 100 (Solarbio, China), blocked for 30 min with goat serum (Booster, China), and incubated with primary antibodies against mTOR (1:1000, Cell Signaling Technology #2983, USA) or RUNX2 (1:1000, Cell Signaling Technology #12556s, USA) overnight at 4 °C. Then, they were incubated with secondary antibody DyLight 488 (1:100, Abbkine #A23220, USA) for one hour. In addition, the nuclei were stained with DAPI, and actin was stained with TRITC-phalloidin (Solarbio, China). Finally, samples were observed by a Spinning Disc Laser Confocal Microscope (Dragonfly 200, Oxford Instruments Andor, USA). Under the condition that the shooting parameters were unchanged, the fluorescence intensity of related proteins was used to indicate the relative expression of protein in the cells.

The lymphocytes in peripheral blood were isolated by discontinuous Percoll gradient density (1.020–1.070 g/ml) on the 1st, 7th, and 14th day, and at weeks 3, 5, and 7. The lymphocytes were adjusted to 1 × 10⁶ cells/ml and incubated with anti-CD4 monoclonal antibodies (mixed with CD4-1 and CD4-2, diluted 1:1,000) and anti-IgM monoclonal antibodies (diluted 1:1,000). Then, the cells were stained with Dylight 488 (Abbkine) mouse IgG antibody at 37°C for 1 h. Flow cytometry was performed using FACSCalibur (BD Biosciences) flow cytometers and analyzed by FlowJo 10.7 (TreeStar).

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).

Cells were seeded on slide covers in 24‐well plates, grown to 80% confluence, and treated as described in 2.10. Then, the cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS three times (10 minutes each), permeabilized with 0.25%Triton X‐100 in 3% BSA for 10 minutes, washed with PBS three times, then blocked in 5% BSA at room temperature for 40 minutes. After washing with PBS, the cells were incubated with antibodies against LC3A/B (1:250, Affinity), AMPK (1:200, Affinity), SKP2 (1:200, Affinity), CARM1 (1:250, Affinity) and H3R17me3a (1:200, Affinity) at 4°C overnight. Then, the cells were washed with PBS and incubated with a fluorescent secondary antibody (DyLight 594, E032420, EarthOx, USA; DyLight 488, A23220, Abbkine) in the dark for 60 minutes, stained with DAPI for 10 minutes, then washed twice with PBS. Images were captured using a fluorescence microscope (Eclipse Ti‐S, Nikon, Japan).

Cells were seeded in 24-well plates (3000 cells/well) overnight. For phosphorylated (p)-STAT3 staining, cells were treated with IL-6 (25 ng/ml) for 24 h (37 °C). Then, 4% paraformaldehyde was used to fix the cells for 20 min at room temperature, followed by incubation with 50 µl of normal goat serum (cat. no. AR0009, BOSTER Biological Technology Co., Ltd.) for 30 min at room temperature. The primary antibodies p-STAT3^Y705 (1:100; cat. no. 9145, Cell Signaling Technology, Inc.), gp130 (1:100; cat. no. MAB4681-SP, Novus Biologicals, LLC) and ART1 (1:100; cat. no. ab185293, Abcam) were added for incubation at 4 °C overnight, after which the fluorescent secondary antibody (red light, DyLight 549, 1:100, cat. no. A23340, Abbkine Scientific Co., Ltd.; and green light, DyLight 488, 1:100, cat. no. A23220, Abbkine Scientific Co., Ltd.) was added for incubation at room temperature for 1 h. DAPI staining was then conducted for 5 min. Images were captured using an AMG EVOS FL microscope. The average optical density of p-STAT3 and gp130, together with the colocalization rate of gp130 and ART1, were determined from three randomly selected images using ImageJ software (version 1.53a, National Institutes of Health). The Pearson correlation and overlapping coefficient results from ≥ 30 cells from three independent experiments are shown as a bar graph (error bars, SEM).