Dab chromogen kit

For immunohistochemical (IHC) staining, paraffin-embedded HNSCC tissue samples (in vivo) were deparaffinized, rehydrated, and incubated with primary antibodies (1:100 dilutions) overnight at 4°C. The slides were then incubated with a biotin-labeled secondary antibody (1:100 dilutions) for 40 min at 37°C. Cells were visualized using a Vectastain ABC kit and a DAB Chromogen kit (Vector Laboratories, Burlingame, CA, USA). The primary antibodies used in this investigation are listed in Supplementary Table 1.
For immunofluorescence staining, HNSCC cells were grown on 18-mm cover glasses and treated with HJC0152 for 24 h. Immunofluorescence staining was conducted with primary antibodies against STAT3, pSTAT3 (Tyr705) (1:100 dilutions, Abcam, Cambridge , UK), and β-catenin (1:100 dilutions, Cell Signaling Technology, Danvers, Massachusetts, USA). The cells were then washed with PBS and incubated with Alexa Fluor 488 or Alexa Fluor 594 secondary antibodies (Cell Signaling Technology). The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; ThermoFisher Scientific), and each slide was visualized using an FV-1000 laser scanning confocal microscope (Olympus, Ishikawa, Japan).