Pv 6001 18

Immunostaining was performed using a rabbit two-step immunohistochemistry kit (Zsbio, PV6001). Briefly, the samples were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. The sections were then baked for 1 h at 65°C, deparaffinized in an alcohol series, and subjected to heat-induced epitope recovery (Zsbio, ZLI-9065). Subsequently, the sections were incubated with FOSL1 antibodies (Abcam, AB124722) and secondary biotinylated antibody (Zsbio, PV-6001-18). In this study, 3,3’-diaminobenzidine (DAB) was used to detect the immunoreactions. The immunohistochemistry (IHC) score of each sample was assessed by the staining intensity and the percentage of positive cells, and it was evaluated by the following formula: 1 × percentage of cells staining weakly + 2 × percentage of cells staining moderately + 3 × percentage of cells staining strongly.

The ameloblastoma samples were obtained from Guanghua School of Stomatology, Sun Yat-sen University. The tumors were fixed in 4% paraformaldehyde for 24 h, dehydrated, embedded in paraffin, and sectioned at 4 μm thickness. After dewaxing, the sections were subjected to heat-induced epitope recovery (Zsbio, Cat#ZLI-9065) and incubated in 3% H₂O₂ for 10 minutes. Then, the sections were washed and incubated with primary antibodies against KI67 (Novus, Cat#NB500-170; 1:200) and EZH2 (Cell Signaling Technology, Cat#5246 S; 1:200) overnight at 4 °C. After incubation with the secondary biotinylated antibody (Zsbio, Cat#PV-6001-18) for 40 minutes, horseradish peroxidase conjugation was carried out with a DAB kit (Zsbio, Cat#ZLI-9017). The scores of each sample were assessed via the staining intensity and area of tumor cells as described previously.^{25 (link)} For the H&E assay, paraffin-embedded tumor sections were stained with an H&E kit (Solarbio, Cat#G1120-100) following the manufacturer’s instructions.