Dylight 594

Cells were fixed using 4% formaldehyde and permeabilized using 0.5% Triton X-100 (BioFroxx, Kronbergim Taunus, Germany). Samples were blocked using 5% bovine serum albumin (ComWin Biotech, Beijing, China) and incubated with primary antibody at 4 °C overnight, and a secondary antibody, DyLight 594 (Abbkine, Wuhan, China), for 1 h. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) for 10 min. Images were captured during confocal microscopy.

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.