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.
Orb10573
Manufactured by Biorbyt
Orb10573 is a lab equipment product from Biorbyt. It is designed for use in scientific research and laboratory settings.
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6 protocols using orb10573
1
Immunohistochemistry of Xenograft and Transgenic Mice
2
CRISPR-Induced Protein Expression Analysis
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After 48 h of transfection of the CRISPR plasmid, the protein of the cells was extracted and quantified. Forty micrograms of total proteins were used in the 1% SDS-PAGE electrophoresis. The primary antibodies used were rabbit anti-HPV16-E7 (1:200, orb10573, Biorbyt), rabbit anti-RB (1:1,000,10048-2-Ig, Proteintech), rabbit anti-CDK2 (1:200,10122-1-AP, Proteintech), rabbit anti-E2F1 (1:200, 12171-1-AP, Proteintech), rabbit anti-GAPDH (1:5,000, 60004-1-Ig, Proteintech). The experiment was repeated 3 times.
3
Immunohistochemical Analysis of HPV-Related Markers
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Paraffin-embedded sections (5 μm) were subjected to H&E staining and immunohistochemical (IHC) staining. The slides were incubated overnight at 4°C with rabbit 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-CD34 (1:100, BA0532, Wuhan Boster Bio-Engineering), rabbit anti-CDK2 (1:400, ab6538, Abcam), and rabbit anti-E2F1 (1:200, 12171-1-Ap, Proteintech) as primary antibodies. Antibody detection was performed using diaminobenzidine (DAB). Images were photographed from three randomly chosen fields using cellSens Dimension (version 1.8.1, Olympus). To quantitatively analyze the expression level, the staining score was determined according to the staining depth and the ratio of positive stained positive cells. Dye depth: 0 is no dye, 1 is light yellow, 3 is brown-yellow, 2 is between 1 and 3. The proportion of positive stained positive cells is calculated to the total number of cells. Staining score = staining depth × positive stained cell ratio.
4
Immunohistochemical Analysis of HPV16 E7 and Cell Cycle Markers
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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.
5
Immunohistochemical Analysis of HPV16E7 and RB
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The xenografts in nude mice and the uterine cervixes and vaginas of the transgenic mice were isolated and fixed by 4% paraformaldehyde. Paraffinembedded 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℃ by the following primary antibodies: rabbit anti-HPV16E7 (1:100, orb10573, Biorbyt), rabbit anti-RB
6
CRISPR Transfection Protein Analysis
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After 48 h of transfection of the CRISPR plasmid, the protein of the cells was extracted and quantified. Forty micrograms of total proteins were used in the 1% SDS-PAGE electrophoresis. The primary antibodies used were rabbit anti-HPV16-E7 (1:200, orb10573, Biorbyt), rabbit anti-RB (1:1,000,10048-2-Ig, Proteintech), rabbit anti-CDK2 (1:200,10122-1-AP, Proteintech), rabbit anti-E2F1 (1:200, 12171-1-AP, Proteintech), rabbit anti-GAPDH (1:5,000, 60004-1-Ig, Proteintech).
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