Ki67 antibody

Manufactured by Vector Laboratories

Sourced in United States

The Ki67 antibody is a laboratory reagent used to detect the presence of the Ki67 protein, a cellular marker associated with cell proliferation. The Ki67 antibody can be used in various immunohistochemical and flow cytometry applications to identify and quantify proliferating cells.

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Lab products found in correlation

Cf633 conjugated goat anti mouse igg secondary antibody, by Biotium (2 mentions) Superfrost plus slide, by Thermo Fisher Scientific (2 mentions) Hoechst stain solution, by Merck Group (1 mentions) Gemcitabine hydrochloride, by Merck Group (1 mentions) Matrigel matrix, by Corning (1 mentions) Dcx antibody, by Santa Cruz Biotechnology (1 mentions) Tbr2 antibody, by Abcam (1 mentions) Neurod, by Santa Cruz Biotechnology (1 mentions) Bx41 upright microscope, by Olympus (1 mentions) Tmr red in situ cell death detection kit, by Roche (1 mentions) 3 3 diaminobenzidine (dab), by Merck Group (1 mentions) Alexa fluor 594 anti rabbit, by Thermo Fisher Scientific (1 mentions) Canto flow cytometer, by FlowJo (1 mentions) Foxp3 fixation permeabilization kit, by Thermo Fisher Scientific (1 mentions) Total caspase 3, by Cell Signaling Technology (1 mentions) Total erk, by Cell Signaling Technology (1 mentions) Cm h2dcfda, by Thermo Fisher Scientific (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

Mouse anti-Ki67 antibody Rabbit anti-Ki67 antibody

10 protocols using ki67 antibody

Multicolor Flow Cytometry Protocol

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SPL-specific antibody has been described previously (Borowsky et al., 2012 (link); Newbigging et al., 2013 (link)). Actin antibody and Hoechst stain solution were procured from Sigma-Aldrich. Fluorochrome- or biotin-conjugated monoclonal antibodies, CD4-efluor 450 (clone RM4-5), CD4-FITC (clone RM4-4), CD8-FITC (clone 53-6.7), CD8-PE (clone 53-6.7), CD62L-PerCP-Cy5.5 (clone MEL-14), CD69-PE-Cy7 (clone H1.2F3), B220-APC (clone RA3-6B2), CD45.1-PE (clone A20), CD45.2-APC (clone 104), CD45RB-APC (clone C363.16A), and CD11c-biotin (clone N4118) were from eBioscience. Antibodies against CK8 (clone TROMA-1-C) and CD31 (clone 2H8-C) were from Developmental Studies Hybridoma Bank. F4/80 antibody (clone A3-1) and CD45-FITC (clone YW62.3) were from AbD Serotec. Ki67 antibody was from Vector Laboratories.

Zamora-Pineda J., Kumar A., Suh J.H., Zhang M, & Saba J.D. (2016). Dendritic cell sphingosine-1-phosphate lyase regulates thymic egress. The Journal of Experimental Medicine, 213(12), 2773-2791.

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Piperlongumine and Gemcitabine Synergy

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Piperlongumine (PL) was purchased from Indofine Chemical Company. Gemcitabine hydrochloride (GEM) was purchased from Sigma-Aldrich. PL and GEM were dissolved in 100% DMSO at stock concentrations of 100 mM and diluted in medium to working concentrations. Matrigel matrix was obtained from Corning. DCFDA was purchased from Life Technologies. Ki-67 antibody was purchased from Vector Labs. CF633-conjugated goat anti-mouse IgG secondary antibody was obtained from Biotium.

Mohammad J., Dhillon H., Chikara S., Mamidi S., Sreedasyam A., Chittem K., Orr M., Wilkinson J.C, & Reindl K.M. (2017). Piperlongumine potentiates the effects of gemcitabine in in vitro and in vivo human pancreatic cancer models. Oncotarget, 9(12), 10457-10469.

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Quantifying Neurogenesis in Dentate Gyrus

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Dissected brains were fixed in 4% paraformaldehyde overnight, following dehydration in 25% sucrose in phosphate-buffered saline (PBS). All sections for immunohistochemistry were sliced at a thickness of 40 µm. Brain sections were mounted on SuperFrost Plus slides (Thermo) and dried overnight. Slides were then incubated in 0.01 M citric acid buffer for 20 min at 95 °C, 3% H₂O₂ for 10 min, rinsed in PBS, and incubated overnight at room temperature in DCX antibody (1:250, Santa Cruz), Ki67 antibody (1:1,000, Vector Laboratory), Tbr2 antibody (1:1,000, Abcam), and NeuroD (1:1,000, Santa Cruz). Subsequently, we used a standard IgG ABC kit (Vector Laboratory) procedure according to the manufacturer’s instructions and incubated the slides for 5 to 10 min with a Sigma DAB (3,3′-diaminobenzidine) tablet.
For quantification, all slides were randomized and coded before quantitative analysis. Slides (half-brain) were examined under a 20× objective. Labeled cells were counted on every eighth section through the entire rostrocaudal extent of the granule cell layer (six sections per animal). The number of cells counted was then multiplied by 16 to obtain an estimate of the total number of positive cells in the dentate gyrus, as previously described (37 (link)).

Chen Y.J., Deng S.M., Chen H.W., Tsao C.H., Chen W.T., Cheng S.J., Huang H.S., Tan B.C., Matzuk M.M., Flint J, & Huang G.J. (2021). Follistatin mediates learning and synaptic plasticity via regulation of Asic4 expression in the hippocampus. Proceedings of the National Academy of Sciences of the United States of America, 118(39), e2109040118.

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Fibroblast Proliferation on 2D and 3D Substrates

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To confirm that the effects of substrate rigidity on the number of Ki67⁺ fibroblasts were comparable for 2D films and 3D scaffolds, fibroblasts (10⁵/film) isolated from the granulation tissue were cultured on 2D-5, 2D-24, and 2D-266 films cast in 12-well tissue culture plates. There were no statistical differences in the number of cells attached to each film at 16 hours post-seeding as determined by a total protein assay. After 3 days of culture, cells were fixed in 4% Formaldehyde and blocked in Odyssey blocking buffer (PBS) (diluted one time in PBS-T) for 1 h. For Ki67 detection, fixed cells were incubated with Ki67 antibody (Vector, 1:1000) at room temperature for 1 h, and an Alexa Fluor^® 488-tagged secondary antibody was applied. Images were obtained with a 20x objective on an Olympus BX41 upright microscope. The number of positively stained cells at day 3 was measured for each film from 6 fields of view observed with a 40X objective.

Guo R., Merkel A., Sterling J., Davidson J, & Guelcher S. (2015). Substrate Modulus of 3D-Printed Scaffolds Regulates the Regenerative Response in Subcutaneous Implants through the Macrophage Phenotype and Wnt Signaling. Biomaterials, 73, 85-95.

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Lung Tissue Analysis of Cellular Dynamics

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Lungs were collected from animals and fixed overnight in 10% formalin supplied with 1mM NaF, 1mM NaVO3, 1mM PMSF. Hematoxylin and eosin (H&E) staining and Immuno-histological (IHC) staining were performed on 5μm formalin-fixed paraffin sections. The antibodies used were described in Western Blotting section. The ABC-Elite Rabbit IgG kit (Vector Laboratories) was used as secondary detection system (45min incubation at room temperature). DAB (Sigma) was used as a substrate. Ki-67 antibody (Vector Laboratories) was used to assess the proliferation of cells. Apoptosis was measured by TUNEL assay with TMR red In Situ Cell Death detection Kit (Roche) according to the manual. The nucleus was stained with DAPI.

Hu Z., Hu Y., Liu X., Xi R., Zhang A., Liu D., Xie Q, & Chen L. (2015). Tumor driven by gain-of-function HER2 H878Y mutant is highly sensitive to HER2 inhibitor. Oncotarget, 6(31), 31628-31639.

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Immunohistochemical Analysis of Tumor Samples

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At the termination of the animal experiments, mice were sacrificed, and the tumor tissues were preserved in 10% formalin. Tissue sections (5 µm) were prepared and processed for immunohistochemistry as described previously (8 (link)). The following antibodies were used for IHC, Ki-67 antibody (Vector laboratories, USA) and MDA-7 (IL-24) antibody (Genhunter, Nashville, TN, USA). Images were analyzed microscopically at a magnification of 40X.

Pradhan A.K., Bhoopathi P., Maji S., Kumar A., Guo C., Mannangatti P., Li J., Wang X.Y., Sarkar D., Emdad L., Das S.K, & Fisher P.B. (2022). Enhanced Cancer Therapy Using an Engineered Designer Cytokine Alone and in Combination With an Immune Checkpoint Inhibitor. Frontiers in Oncology, 12, 812560.

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Assessing KDM5B Reporter Activity

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Melanoma cells stably expressing the KDM5B reporter were treated with indicated doses of inhibitors for 72 hours. Cells were trypsinized, washed in PBS and resuspended in PBS containing 0.5 g/mL live cell stain 7-AAD (Biolegend, San Diego, CA) for FACS analysis using a Canto Flow Cytometer. Upon exclusion of doublets and dead cells, the KDM5BGFP-positive population was determined by comparison to parental, untransduced cells.
For antibody staining, KDM5B reporter cells were permeabilized with Foxp3 Fixation/Permeabilization kit (eBioscience, San Diego, CA) and stained with Ki67 antibody (Vector Laboratories, Burlingame, CA; product number VP-K451). Anti-rabbit AlexaFluor 594 (ThermoScientific) was used as secondary antibody. Cells were acquired on a Canto Flow Cytometer and data was analyzed using FlowJo software.

Trousil S., Chen S., Mu C., Shaw F.M., Yao Z., Ran Y., Shakuntala T., Merghoub T., Manstein D., Rosen N., Cantley L.C., Zippin J.H, & Zheng B. (2017). Phenformin enhances the efficacy of ERK inhibition in NF1-mutant melanoma. The Journal of investigative dermatology, 137(5), 1135-1143.

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Puromycin Cytotoxicity Assay Protocol

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Puromycin was purchased from Sigma-Aldrich, St. Louis, MO, USA. A CellTiter-Glo^® luminescent cell viability assay kit was purchased from Promega, Madison, WI, USA. Ki67 antibody was purchased from Vector Labs, Burlingame, CA, USA. GSTP1 antibody was obtained from Santa Cruz Biotechnology, Dallas, TX, USA. Antibodies to GAPDH, β-actin, phospho-JNK (Thr 183/Tyr 185), total JNK, p65, pERK, total ERK, cleaved caspase-3, total caspase-3, phospho-c-Jun (Ser 73), total c-Jun, and Sp1 were obtained from Cell Signaling Technology, Danvers, MA, USA. Horseradish peroxidase (HRP)-linked anti-mouse and anti-rabbit IgG secondary antibodies were obtained from Cell Signaling Technology. CF633-conjugated goat anti-mouse IgG secondary antibody was obtained from Biotium, Fremont, CA, USA. CM-H₂DCFDA was purchased from Life Technologies, Carlsbad, CA, USA. Glutathione was purchased from Calbiochem, Burlington, MA, USA.

Singh R.R., Mohammad J., Orr M, & Reindl K.M. (2020). Glutathione S-Transferase pi-1 Knockdown Reduces Pancreatic Ductal Adenocarcinoma Growth by Activating Oxidative Stress Response Pathways. Cancers, 12(6), 1501.

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Antibody Panel for Cell Signaling Analysis

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Rabbit polyclonal antibodies to GFP, phosphorylated ERK1/2, phosphorylated RB, ERK1/2, cyclin D1, phosphorylated FADD at Ser¹⁹⁴, AURKA, phosphorylated β-Catenin, β-Catenin, phosphorylated MDM2, and PLK1 were purchased from Cell Signaling Technology. The antibody for human FADD was obtained from BD Biosciences. Antibodies against cyclin B1, CK1α, MDM2 and p53 were purchased from Santa Cruz Biotechnology. The antibody against mouse FADD used for western blotting was initially purchased from Epitomics (cat# 3523-1), and then from Abcam (cat# ab124812). Antibodies for BUB1, β-Actin, mouse FADD (cat# ab24533) and GFP were from Abcam. Ki-67 antibody was obtained from Vector Labs. CKI-7 was from Sigma. PD0325901 was purchased from Selleck, and Lonafarnib was bought from Cayman Chemical. Alamar Blue was purchased from Invitrogen. Luciferin was obtained from Promega. Adenovirus-Cre was bought from the University of Michigan Vector core.

Bowman B.M., Sebolt K.A., Hoff B.A., Boes J.L., Daniels D.L., Heist K.A., Galbán C.J., Patel R.M., Zhang J., Beer D.G., Ross B.D., Rehemtulla A, & Galbán S. (2015). Phosphorylation of FADD by the kinase CK1α promotes KRASG12D-induced lung cancer. Science signaling, 8(361), ra9.

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Immunohistochemical Evaluation of Ki-67

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Collected tissues were processed and sectioned as reported previously [13, 14] . Sections were deparaffinized, rehydrated, and subjected to antigen retrieval (Vector Laboratories) treatment for 20 min followed by blocking the endogenous peroxidase with 3% hydrogen peroxide in methanol for 20 min. Horse serum (Vector Laboratories) was used for blocking the sections for 30 min at room temperature and incubated with Ki-67 antibody (#VP-RM04; Vector Laboratories, 1:100) overnight at 4°C. Sections were washed twice with 1x PBS and treated with biotinylated anti-rabbit/mouse IgG secondary antibody (Vector Laboratories) for 45 minutes, followed by ABC reagent for 30 min. Diaminobenzidine (Vector Laboratories) was used as a substrate to develop the stain. Hematoxylin was used as a counterstain followed by dehydration with alcohol, clearing with xylene, and mounting with permanent mounting media (Vector Laboratories). Stained sections were observed under the microscope (Leica DM500), and images were taken at different magnifications.

Suresh V., Mohanty V., Avula K., Ghosh A., Singh B., Reddy R.R., Suryawanshi A.R., Raghav S.K., Chattopadhyay S., Prasad P., Swain R., Dash R., Parida A., Syed G.H., & Senapati S. (2021). Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host-factors having implication in the disease pathogenesis and severity.

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