After deparaffinization, rehydration, antigen retrieval, and blockade of endogenous peroxidase, tissues were incubated with anti-TUNEL (Roche), anti-cleaved Caspase 3 (Servicebio), anti-Ki67 (Servicebio), and anti-CD31 (Abways) primary antibodies at 4 °C overnight. Next, secondary antibodies were applied, and diaminobenzidine (DAB) (Dako) solution was used as a chromogen. Finally, the sections were counterstained with hematoxylin (Sigma-Aldrich) to identify nuclei.
Anti ki67
Manufactured by Wuhan Servicebio Technology
Sourced in China, United States
Anti-Ki67 is a laboratory reagent used for the detection and quantification of the Ki67 protein, a well-established marker of cell proliferation. It is commonly used in research and diagnostic applications to assess the proliferative state of cells.
Automatically generated - may contain errors
Lab products found in correlation
Cy3 goat anti rabbit, by Wuhan Servicebio Technology (3 mentions)
Anti caspase 3, by Wuhan Servicebio Technology (3 mentions)
Anti cd31, by Wuhan Servicebio Technology (3 mentions)
Anti pcna, by Wuhan Servicebio Technology (2 mentions)
Anti cd8, by Wuhan Servicebio Technology (2 mentions)
Eclipse c1, by Nikon (2 mentions)
Protease inhibitor, by Boster Bio (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Reactive oxygen species detection kit, by Beyotime (2 mentions)
Tunel reaction solution, by Boster Bio (2 mentions)
Annexin 5 fitc pi apoptosis detection reagent, by BD (2 mentions)
Membrane 2 kd, by Solarbio (2 mentions)
Anti caspase 9, by Wuhan Servicebio Technology (2 mentions)
Cell lysate, by Boster Bio (2 mentions)
Anti p53, by Wuhan Servicebio Technology (2 mentions)
Rpmi 1640 cell culture medium, by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
H e staining kit, by Keygen Biotech (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (2 mentions)
Cell cycle detection kit, by Beyotime (2 mentions)
32 protocols using anti ki67
1
Immunohistochemical Analysis of Apoptosis and Proliferation
2
Immunohistochemical Analysis of Tumor Markers
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Paraffin-embedded tumor tissues were sliced into 4-mm sections, and the series of protein expression was determined by IHC. Briefly, after dewaxing, antigen retrieval, serum blocking, slides were incubated with anti-PCK2 (SAB, College Park, MD, USA), anti-Ki67, anti-MMP-2, anti-Bcl-2 anti-c-Myc, anti-MMP-9 (All from Servicebio, Wuhan, China), and anti-PC antibodies (Proteintech, Rosemont, IL, USA) at 4°C overnight. The subsequent steps were performed using the SABC-AP kit with anti-rabbit IgG (Beyotime, Shanghai, China). The IHC was scored as follows: the average positive intensity in the measurement area is 0, 1, 2, and 3 points. Negative without coloring (0 points). Weak yellowish (1 point). Medium-positive brownish-yellow (2 points). Positive dark brown (3 points). The positive rate of cells in the measurement area is 0, 1, 2, 3, and 4 points. 0%–15% is 0, 6%–25% is 1, 26%–50% is 2, 51%–75% is 3, and >75% is 4. The overall positive score is the positive intensity value × positive cell rate value.
3
Immunohistochemical Analysis of Protein Expression
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The clinical tissue samples used in this study were histopathologically and clinically diagnosed at Ruijin Hospital with patient consent and with approval from the Ethics Committee. This information is presented in Table S2 (Supporting Information). IHC staining was performed with the following antibodies: anti‐BHLHE40 (1:100, Novus), anti‐SREBF1 (1:100, Abcam), anti‐SCD1 (1:100, Abcam), and anti‐Ki67 (1:100, Servicebio) to detect protein expression. The protein expressions of BHLHE40, SREBF1, and SCD were analyzed using the digital pathological image analysis software based on artificial intelligence learning (Servicebio Technology) via tracking, color selection, calculation, and TMA. The analysis formula is H‐SCORE = ∑(pi×i) = (percentage of weak intensity ×1) + (percentage of moderate intensity ×2) + (percentage of strong intensity ×3).
4
Immunohistochemical Analysis of Cancer Markers
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Tissue slices were fixed, embedded, deparaffinized, and blocked. The sections were incubated with anti-FOXQ1 (Proteintech, Cat No. 23718-1-AP), anti-KI67 (Servicebio, Cat No. GB111499), anti-PCNA (Servicebio, Cat No. GB11010), anti-N-cadherin (Cell Signaling Technology, Cat No. 13116), anti-Vimentin (Signalway, Cat No. 33541), and anti-LDHA (Proteintech, Cat No. 66287-1-Ig) antibodies at 4 °C overnight. After washing with phosphate-buffered saline (PBS), the sections were incubated in a secondary antibody (Proteintech, Cat No. PR30009) for 1 h at room temperature. Hematoxylin re-staining, imaging, and DAB staining were then performed using standard procedures. Two different pathologists independently assessed the findings.
5
Immunofluorescence Analysis of Mouse Uterine Sections
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Sections of mouse uterus were fixed in precooled acetone and incubated with 0.3% hydrogen peroxide in 10% methanol for 15 min. After washing with PBS, the sections were blocked using an avidin/biotin blocking kit (Vector Laboratories, CA, USA). After incubation, blocking was performed using the Mouse on Mouse Fluorescein Kit (Vector Laboratories), following the manufacturer's instructions. Sections were incubated with anti-Ki-67 (1 : 300, Servicebio), anti-terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (1 : 300, Elabscience), anti-E-cadherin (1 : 300, CST), and anti-vimentin (1 : 300, CST) overnight. In the next day, sections were incubated with rabbit anti-mouse IgG for 1 h. After DAPI staining, sections were observed with a confocal microscope (Carl Zeiss LSM800, Prenzlauer, Berlin, Germany). The average fluorescence intensity of immunofluorescence was measured using image analysis software (ZEN, Zeiss, Germany) and quantitatively analyzed.
6
Immunostaining of Mouse and Human Skin
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Cryosections (10 μm) of mouse skin were cut using a cryostat (Leica, CM1950) and fixed in ice-cold acetone for 15 min. Human and mouse paraffin sections (6 μm) were dewaxed and antigen unmasked. The slices were blocked with blocking buffer(5% normal goat serum, 3% BSA, and 0.2% Triton X-100 in PBS) for 1 h and stained with anti-Cochlin (Beyotime Biotechnology, cat: AF6522, 1:100), anti-Tenascin C (Abcam, cat: ab10893, 1:100), anti-Ki67 (Servicebio Technology, cat: GB121141, 1:500) and anti-β-III tubulin (Abcam, cat: ab78078, 1:1000; ab18207, 1:100) primary antibodies overnight at 4 °C. Afterward, the slices were rinsed three times in PBS and stained with relative secondary antibodies (all from Invitrogen) for 1 h at RT. Nuclei were stained with DAPI (BD Biosciences, cat: 564907) at RT for 5 min. After being washed in PBS, the slices were mounted (Beyotime Biotechnology, cat: P0126) and visualized under a confocal microscopy system (Leica, TSC SP8).
7
Quantifying Ki-67 Expression in Tissues
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Immunohistochemistry was performed as previously reported. Briefly, frozen tissue sections were fixed and antigenically repaired. Afterward, tissue sections were incubated overnight at 4 °C with a mouse monoclonal anti-Ki-67 (1:100; Servicebio, Wuhan, China) followed by a secondary antibody. After rinsing in water, sections were counterstained with hematoxylin, dehydrated, and capped. Afterward, they were observed under a microscope and quantified by Image J software and the IHC Profiler plug-in.
8
Immunohistochemical Staining of Tumor Samples
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As previously mentioned, immunohistochemical (IHC) staining was carried out. To summarize, 4 μm thick slices of paraffin-embedded tumor samples from the mouse model experiment and clinical ESCC tissues were cut. After deparaffinization, the sections were rehydrated. Following antigen retrieval, the slices were incubated overnight at 4 °C with anti-Nur77 (1:200, Cat# NB100-56745, Novus), anti-IRF1 (1:100, Cat# 11335-1-AP, Proteintech), anti-PD-L1 (1:5000, Cat# 66248-1-lg, Proteintech), anti-Ki67 (1:300, Cat# GB121141, Servicebio) anti-PCNA (1:1000, Cat# GB12010, Servicebio) and anti-CD8 (1:400, Cat# GB15068, Servicebio) antibodies. Following multiple washes, the sections were incubated for 1 h at room temperature with an HRP-conjugated secondary antibody and stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Servicebio, Wuhan, China). The slides were imaged using a microscope (Olympus BX43F, Japan), and the images were processed using CaseViewer software.
9
Immunohistochemical Analysis of Tissue Samples
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The tissue samples were embedded in paraffin and cut into 3 μm sections, and immunohistochemical staining was performed to detect specific markers using the following antibodies (obtained from Servicebio): anti-cleaved caspase-3 (Catalog no. GB13436), anti-Ki67 (Catalog no. GB111499), and anti-CD68 (Catalog no. GB113150). The stained slides were scanned using the 3D HISTECH system. Positively stained cells on each slide were identified using the artificial intelligence image analysis software Aipathwell (Service Bio). The staining index (%) was calculated as the percentage of positive cells out of the total number of cells. The log2 fold change (infected monkeys vs NC) in the staining index was plotted as a heatmap using Graph Pad Prism 8.0.
10
Quantification of Tumor Cell Proliferation
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After resection, the tumor tissues were fixed in 4% paraformaldehyde overnight. Then, the fixed tumor tissues were embedded with paraffin and sectioned at 5 μm thickness and then stained with anti-Ki67 (ServiceBio #GB111499). Pictures were taken with the Olympus VS120 imaging system, and representative pictures were presented in the manuscript. The percentage of proliferative tumor cells was quantified by the ratio of Ki67-positive tumor cells to total tumor cells in the tumor tissues.
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