Ab6538

The xenografts in nude mice and the uterine cervixes and vaginas of the transgenic mice were isolated and fixed by 4% paraformaldehyde. Paraffin-embedded sections (5 μm) were subjected to IHC staining according to the Proteintech protocol (http://www.ptgcn.com/support/protocols/). After antigen retrieval for 30 min and blocking with 3% hydrogen peroxide for 20 min, the paraffin sections were incubated overnight at 4 °C by the following primary antibodies: rabbit anti-HPV16E7 (1:100, orb10573, Biorbyt), rabbit anti-RB (1:200,10048-2-Ig, Proteintech), rabbit anti-CDK2 (1:200, ab6538, Abcam), rabbit anti-E2F1 (1:200, 12171-1-AP, Proteintech), rabbit anti-Ki67 (1:200, ab16667, Abcam), rabbit anti-p16 (1:100, A11337, Abclonal), rabbit anti-PCNA (1:100, 10205-2-AP, Proteintech), rabbit anti-CD31 (1:100, 11265-1-AP, Proteintech), rabbit anti-Caspase-3 (1:100, 19677-1-AP, Proteintech). Next, samples were incubated with proper secondary antibodies for 1 h at room temperature. A 3,3’-diaminobenzidine (DAB) kit was used to detect the antibodies, and the slide was photographed at the random site using the cellSens Dimension (version 1.8.1, Olympus). The staining intensity was measured by ImagePro Plus.

Paraffin-embedded sections (5 μm) were stained with H and E and immunohistochemical (IHC) staining. The slides were incubated overnight at 4°C with mouse anti-HPV16 E7 (1:50, orb10573, Biorbyt), rabbit anti-P16 (1:100, A0262, Abclonal), rabbit anti-RB1 (1:100, 10048-2-Ig, Proteintech), rabbit anti-Ki67 (1:100, ab16667, Abcam), rabbit anti-CDK2 (1:400, ab6538, Abcam), rabbit anti-E2F1 (1:200,12171-1-Ap, Proteintech), rabbit anti-TP53 (1:150, A16989, Abclonal), rabbit anti-14-3-3 Sigma (1:50, A1026, Abclonal) and rabbit anti-P21 (1:150, A11454, Abclonal) primary antibodies. Diaminobenzidine (DAB) was used for antibody detection. Images were photographed from three randomly selected fields using cellSens Dimension (version 1.8.1, Olympus). Staining was assigned a score using a semiquantitative six category grading system: 0, no staining; 1,1%to 10% staining; 2, 11% to 25% staining; 3, 26% to 50% staining; 4, 51% to 75% staining; and 5, >75% staining. Staining intensity was assigned a score using a semiquantitative four-category grading system: 0, no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. Every core was assessed individually and the mean of three readings was calculated for every slide. The staining score was determined separately by two experts under the same conditions.