Anti ncam1

Total protein was extracted from tissues and was measured using a BCA kit. Then, protein samples were transferred onto PVDF membranes. The membranes were blocked for 1 h at 37°C. After that, the membranes were incubated overnight with the primary antibodies anti-NCAM1 (1: 2000; Abcam, UK) and anti-β-actin (1: 1000; Abcam, UK) at 4°C, followed by addition of secondary antibody (1: 5000; Abcam, UK) at room temperature for 1 h. Finally, the protein blots were visualized using an infrared fluorescence scanning imaging system.

Tissue sections were dewaxed and dehydrated. Then, 3% hydrogen peroxide was used to eliminate endogenous peroxidase activity. The sections were blocked with 5% goat serum and incubated for approximately 10 min. The sections were incubated overnight with the primary antibodies anti-NCAM1 (1: 200; Abcam, UK) and anti-E-cadherin (1: 200; Abcam, UK) at 4°C, followed by incubation with biotinylated secondary antibody at room temperature for 20 min. Finally, using the DAB kit, the sections were rinsed, counterstained, dehydrated, cleared, and sealed. In the negative control group, PBS was used instead of the primary antibody. The semiquantitative scores were assessed based on staining intensity and the percentage of positive cells. Three fields of vision were selected for scoring at 200 magnification and the scores were averaged. The scoring criteria were: strong positive (4 points), positive (3 points), weak positive (2 points), and negative (1 point).