Ab233391

At the end of the study, the tumor tissues were removed, fixed in 4% paraformaldehyde overnight, embedded in paraffin, and cut into 4 μm-thick cross sections. After deparaffinization, the paraffin sections were subjected to antigen retrieval with citrate buffer and treated with 3% H₂O₂ for 10 min at room temperature. The sections were then blocked with 5% non-immune goat serum for 30 min at 37°C and incubated with the corresponding primary antibodies at 4°C overnight. The primary antibodies used were rabbit anti-fascin antibody (1:1000, Abcam, ab126772) and rabbit anti-PTK6 antibody (1:1000, Abcam, ab233391). Horseradish peroxidase-conjugated secondary antibodies (PV-9001, ZSGB-Bio, Beijing, China) were added the next day for 30 min at 37°C, and a DAB kit (ZLI-9018, ZSGB-Bio) was used for color development according to the manufacturer’s instructions. Hematoxylin was used to counterstain the nuclei during IHC staining. Sections were incubated with non-immune IgG secondary antibody only, and no primary or secondary antibodies were used as negative controls.

Proteins were extracted from cells or fresh tissue lysates from the treatment and experimental groups. Protein extracts were prepared in RIPA lysis buffer containing PMSF (100:1). A BCA protein assay kit (Cwbiotech, Beijing, China) was used to determine the protein concentration. Protein extracts were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, MA, United States ), blocked with 5% BSA for 1 h at room temperature, and then incubated with the corresponding primary antibodies overnight. Finally, the proteins were visualized using chemiluminescent horseradish peroxidase substrate (Millipore). The levels of target proteins, such as β-actin, were normalized to those of the loading control. Six independent experiments were performed to derive the mean values for statistical analysis. The primary antibodies used were rabbit anti-fascin antibody (1:1000, Abcam, Cambridge, UK, ab126772), rabbit anti-PTK6 antibody (1:1000, Abcam, ab233391), and rabbit anti-GAPDH antibody (1:1000, Cell Signaling Technology, MA, United States , 2118).