Anti ki67

Manufactured by Cell Marque

Sourced in United States

Anti-Ki67 is a laboratory reagent used for the detection of the Ki-67 protein, which is a well-established marker of cellular proliferation. It is commonly utilized in immunohistochemistry and other analytical techniques to assess the proliferative activity of cells in various tissue samples.

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Rabbit anti-Ki67 (3 citations)

5 protocols using anti ki67

Immunofluorescent Cell Labeling Protocol

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Cells were fixed in 4% formaldehyde for 10 min with shaking at room temperature, permeabilized for 5 min with 0.5% Triton X-100 in PBS, and blocked for 1 h at room temperature with 5% BSA in PBS containing 0.1% Triton X-100. The cells were labeled overnight at 4 °C with the primary antibodies anti-Ki67 (1:200, 275R, Cell Marque) and anti-phosphorylated histone 3 (1:200, 9701, Cell Signaling). After three washes with PBS, samples were labeled for 1 h at room temperature with fluorescent secondary antibodies (Abcam) followed by 10 min of DAPI staining for nuclear visualization. After three washes in PBS, cells were imaged with a Nikon Eclipse Ti2 microscope.

Kastan N., Gnedeva K., Alisch T., Petelski A.A., Huggins D.J., Chiaravalli J., Aharanov A., Shakked A., Tzahor E., Nagiel A., Segil N, & Hudspeth A.J. (2021). Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues. Nature Communications, 12, 3100.

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Immunohistochemical Analysis of Liver Tissue

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Liver sections were cut at 5‐micron‐thick from frozen tissue, fixed with acetone for 5 min at −20°C, blocked with 10% bovine serum albumin for 1 h, and then incubated overnight with anti‐Laminin or anti‐Ki67 (both at 1:100; Cell Marque). For double‐detection of markers, anti‐CD90 (1:50; NovusBio) was incubated overnight, and anti‐CK19 (1:100; Bio SB) was only incubated for 1 h in the same tissue section after removing and washing the first antibody. Primary antibodies were detected using either anti‐mouse (Alexa 488) or antirabbit (Alexa 594), respectively (both at 1:300; Jackson) for 1 h. As an experimental control, the substitution of the primary antibody with either mouse or rabbit isotype control was also included. Images were captured with a microscope AXIO scope (Carl Zeiss). ImageJ plugins¹⁶ were used to quantitatively analyze the positive label in five images per animal and at least three animals per group.

Castro‐Gil M.P., Sánchez‐Rodríguez R., Torres‐Mena J.E., López‐Torres C.D., Quintanar‐Jurado V., Gabiño‐López N.B., Villa‐Treviño S., del‐Pozo‐Jauner L., Arellanes‐Robledo J, & Pérez‐Carreón J.I. (2021). Enrichment of progenitor cells by 2‐acetylaminofluorene accelerates liver carcinogenesis induced by diethylnitrosamine in vivo. Molecular Carcinogenesis, 60(6), 377-390.

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Immunohistochemistry for Tumor Markers

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Immunohistochemistry (IHC) was performed as described (13 (link)). Tumor sections were deparaffinized and antigen retrieval was performed prior to primary antibody incubation. Primary antibodies were anti-survivin (Abcam, ab24479), anti-STAT3 (Abcam, ab325015), and anti-Ki67 (Cell Marque, 275R-18).TUNEL staining was performed on paraffin sections using Apoptag Kit (Millipore, S71003).

Cheng Y., Holloway M.P., Nguyen K., McCauley D., Landesman Y., Kauffman M.G., Shacham S, & Altura R.A. (2014). XPO1 (CRM1) inhibition represses STAT3 activation to drive a survivin-dependent oncogenic switch in triple negative breast cancer. Molecular cancer therapeutics, 13(3), 675-686.

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Immunohistochemical Analysis of Myocardial Tissues

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Myocardial tissues were placed a in tissue-freezing medium (Sakura Finetek USA) and frozen at −20°C. After fixation in 4% polyformaldehyde for 30 min, tissue sections and cultured cells were permeabilized with 1% Triton X-100 in PBS for 5 min and then incubated in 3% BSA for 1 h at room temperature. The samples were subsequently incubated at 4°C overnight with the following primary antibodies diluted in 3% BSA: anti-Ki67 (1:200, 275R; Cell Marque), anti-pH3 (ab170904; Abcam), anti-cardiac troponin T (cTnT; ab33589; Abcam), anti-Aurora B (ab2254; Abcam), anti-α-SMA (ab7817; Abcam), anti-vWF (ab11713; Abcam), and anti-IB4 (ab181548; Abcam). A Click-it EdU Imaging Kit (Life Technologies; #C10638) was used to detect EdU incorporation according to the manufacturer’s instruction. An In Situ Cell Death Detection Kit (Roche, Shanghai, China) was used to detect apoptotic cells in samples. After three washes with PBS, the samples were stained with fluorescent secondary antibodies (Alexa Fluor 488 and/or 594; Abcam) for 1 h at room temperature, followed by 20 min of 4′,6-diamidino-2-phenylindole (DAPI) staining (ab104139; Abcam). Images were captured using confocal laser-scanning microscopy (Carl Zeiss).

Si X., Zheng H., Wei G., Li M., Li W., Wang H., Guo H., Sun J., Li C., Zhong S., Liao W., Liao Y., Huang S, & Bin J. (2020). circRNA Hipk3 Induces Cardiac Regeneration after Myocardial Infarction in Mice by Binding to Notch1 and miR-133a. Molecular Therapy. Nucleic Acids, 21, 636-655.

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Antibody Panel for Signaling Proteins

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Antibodies to detect MET (D1C2), mTOR (7C10), LC3B, PARP, Caspase 3, Cyclin D1, phospho-ALK (Tyr1604), phospho-MET (Tyr1234/1235), AKT, phospho-AKT (Ser473), phospho-MAPK (Tyr202/204), 4EBP1, phospho-4EBP1 (Thr37/46), S6K, phospho-S6K (Ser371), RPS6, phospho-RPS6 (Ser235/236), STAT3, phospho-STAT3 (Tyr705) were from Cell Signaling (Danvers, MA, USA). Anti-RIP3 was from Santa Cruz Biotechnology (Dallas, TX, USA), Anti-Ki-67 from Cell Marque (Rocklin, CA, USA), and Anti-β-Actin from Sigma-Aldrich (München, Germany). ALK-1A4 (immunoblotting) was from Origene (Rockville, MD, USA), ALK-D5F3 (immunohistochemistry) was from Ventana Medical Systems (Tucson, AZ, USA). Crizotinib and rapamycin were purchased from Pfizer (Berlin, Germany) and ChemieTek (Indianapolis, IN, USA), respectively.

Mönch D., Bode-Erdmann S., Kalla J., Sträter J., Schwänen C., Falkenstern-Ge R., Klumpp S., Friedel G., Ott G, & Kalla C. (2018). A subgroup of pleural mesothelioma expresses ALK protein and may be targetable by combined rapamycin and crizotinib therapy. Oncotarget, 9(29), 20781-20794.

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