Goat anti rabbit igg h and l hrp

TBX3 protein expression was detected by immunohistochemical staining according to the instructions of the VECTASTAIN^® Elite^® ABC Kit (Vector Laboratories, USA). First, the paraffin sections were heated at 60°C for 2 h, dewaxed and hydrated sequentially with xylene and ethanol, washed with PBS and double-distilled water, and stained after nuclear antigen retrieval. Secondly, we used TBX3 antibody (1: 300, Abcam, UK) or Ki-67 antibody (1: 300, Abcam, UK) as primary antibody (PBS instead of primary antibody as negative control) and incubated cells overnight at 4 C. Goat Anti-Rabbit IgG H and L (HRP) (Abcam, UK) was incubated as secondary antibody at 37°C for 2 h. Finally, 5 fields per slice were photographed and scored [15 ]: TBX3 protein localized in cytoplasm and Ki67 protein was localized in the nucleus. The cells in the slices were stained yellow (1 point), brown (2 points), and tan (3 points) for positive staining, while the cells that were not stained were scored as negative (0 point). In each field of view, the number of positive cells was scored as: ≤25%=0 points, 25–75%=1 point, and ≥75%=2 points. Results of the staining score multiplied by the percentage score of positive cells was the immunohistochemical score. In this study, TBX3 immunohistochemical score ≥4 was regarded as high expression.