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Anti ki67 antibody ab15580

Manufactured by Abcam
Sourced in United Kingdom

Anti-Ki67 antibody (ab15580) is a mouse monoclonal antibody that recognizes the Ki67 protein, a cellular marker for proliferation. The antibody can be used to detect Ki67 expression in a variety of species and tissues.

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9 protocols using anti ki67 antibody ab15580

1

Immunostaining and Proliferation Assay for Pancreatic Cells

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The cells were fixed with 4% paraformaldehyde in PBS. After blocking with 20% AquaBlock (EastCoast Bio, North Berwick, ME, USA) for 30 min at room temperature, the cells were incubated overnight at 4°C with a guinea pig anti-INS antibody (1:100; Abcam, Tokyo, Japan) or rabbit anti-C-PEPTIDE antibody (1:200; Cell Signaling Technology, Danvers, MA, USA) and then for 1 h at room temperature with fluorescein isothiocyanate (FITC) or Alexa Fluor 647-conjugated anti-guinea pig immunoglobulin G (IgG) (FITC, 1:250 [Abcam] and Alexa Fluor 647, 1:250 [Cell Signaling Technology]), or Alexa Fluor 647-conjugated anti-rabbit IgG (1:250; Cell Signaling Technology). The cells were mounted on slides using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Peterborough, UK). The percentage of INS/C-PEPTIDE-positive cells was calculated based on the ratio of immunostaining-positive cells/DAPI-positive cells in 10 visual fields.
To identify proliferating cells, we used immunohistochemistry (IHC) to detect Ki67 in the nuclei of cells in the G1, S, G2, and M phases of the cell cycle. For this purpose, we used the Histofine Simple Stain MAX PO (R) kit (Nichire Biosciences, Tokyo, Japan) with an anti-Ki67 antibody (ab 15580) (Abcam, Cambridge, UK).
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2

Quantifying Cell Proliferation and Apoptosis

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Frozen sections were fixed in 4% paraformaldehyde in PBS. For Ki67 staining to image cell proliferation, the sections were blocked with blocking solution (0.3% Triton X, 5% horse sera, and 1% goat sera in PBS). Anti-Ki67 antibody (ab15580) (Abcam, Cambridge, MA) was applied to the blocking solution followed by the addition of Alexa Fluor® 488 anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA). To evaluate cell apoptosis, a TUNEL assay was performed, according to the manufacturer's protocol (Promega Corporation, Madison, WI). All images were acquired using a Nikon A1 microscope (Nikon Inc., Melville, NY).
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3

Paraffin Embedding and Immunohistochemistry

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Fixed tumor tissues were processed for paraffin embedding and were sectioned into 4-µm sections. The sections were stained with H&E according to standard protocols. For immunohistochemical staining, the tumor tissues slides were incubated with diluted primary antibodies against anti-Ki67 antibody (ab15580) (Abcam, Cambridge, U.K.) according to the manufacturer’s instructions. The primary antibody was detected using biotinylated goat anti-rabbit antibody followed by 3,3-diaminobenzidine in the kit (Univbio, Shanghai, China).
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4

Immunoblotting Analysis of Signaling Pathways

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The following antibodies were purchased from Cell Signaling Technology: HIF-1α (D1S7W) (Rabbit mAb#36,169); c-Myc (D84C12) (Rabbit mAb #5605); Phospho-Akt (Thr308) (244F9) (Rabbit mAb #4056); Phospho-Akt (Ser473) (587F11) (Mouse mAb #4051); Akt (pan) (11E7) (Rabbit mAb #4685); p70 S6 Kinase (49D7) (Rabbit mAb #2708); Phospho-p70 S6 Kinase (Thr421/Ser424) (Antibody #9204); Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (Rabbit mAb (HRP Conjugate) #8544); p44/42 MAPK (Erk1/2) (137F5) (Rabbit mAb #4695); FAK antibody ( #3285); Phospho-FAK (Tyr576/577) antibody (#3281); CDK4 (D9G3E) (Rabbit mAb #12790); Rb (4H1) (Mouse mAb #9309); Phospho-Rb (Ser807/811) (D20B12) (Rabbit mAb #8516); Cyclin D1 antibody (#2922), PRAS40 antibody (#2610); Phospho-PRAS40 (Thr246) (D4D2) (Rabbit mAb #13175). The followings reagents were purchased from ABCAM: Anti-LOX antibody (ab174316); Anti-TGF beta 1 antibody (ab27969); Anti-Ki67 antibody (ab15580). rLOX was purchased from OriGene Technologies. BAPN was purchased from Sigma-Aldrich.
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5

Baf53a cKO ES Cell Proliferation

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Baf53a cKO ES cells were cultured with or without Tet for 4 days in 24-well plate. Cells were washed by 1 x PBS (−) and fixed by 10% formalin solution for 30 min at room temperature. These cells were washed with 0.2% Triton X-100 in PBS (−) for 3 times and then incubated with 5% FBS-0.2% Triton X-100 in PBS (−) at 37 °C for 1 hour. The cells were then incubated with 50-fold diluted anti-Ki67 antibody (ab15580, Abcam, Cambridge, UK) at 4 °C for overnight, followed by incubation with 100-fold diluted goat anti-rabbit IgG FITC (Santa Cruz Biotechnology). Vectashield mounting medium (Vector Laboratories, Inc. Burlingame, CA) was used for DAPI staining.
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6

In vivo Tumor Formation Assay

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In vivo tumorigenic studies in mice were performed as described previously (Alaoui-Jamali et al., 2009 (link)). In brief, A375 melanoma cells stably expressing miR-612 or together with Espin (1 × 106 cells per mouse) were subcutaneously implanted into the flanks of nude mice (5-week old; Shanghai Laboratory Animal Center, Chinese Academy of Sciences, China). Tumor volume was calculated weekly for 4 weeks, and growth curves were plotted. After the last measurement, the animals were killed. Xenograft tumors were fixed, sectioned, and stained with anti-Ki-67 antibody (ab15580, Abcam, Cambridge, UK; 1:200 dilution), as described previously (Waters et al., 2015 (link)). Animal experiments were approved by the Animal Care and Use Committee of Shandong University.
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7

Histological Analysis of Cell Proliferation

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For HE, sections were stained with hematoxylin and eosin. For IHC, rehydrated sections were staining according to published methods [47 (link)]. Images were captured with a Leica DM2000 microscope. The following antibodies and dilutions were used: anti-Ki67 antibody (ab15580, 1:2000) and goat anti-rabbit IgG H&L (HRP) (ab6721, 1:500) were purchased from Abcam (Cambridge, UK).
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8

Antibody Panel for Cellular Analysis

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The anti-MAP1LC3-I/II antibody (4599 and 3868), anti-lamin (4777) and anti-caspase-3 antibody (9665) were from Cell Signaling, and the anti-SQSTM1 (p62/SQSTM1) antibody (sc-28359), anti-RRM1 antibody (sc-11733) and anti-RRM2 antibody (sc-10844) were from Santa Cruz. The anti-Ki67 antibody (ab15580) was from Abcam, and the anti-p53R2 antibody (600-401-B67) was from Rockland. The anti-γH2AX antibody (05-636) and anti-actin antibody (MAB1501R) were from Millipore.
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9

Umbelliferone Treatment Regulates Cellular Proteins

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Cultured cells were lysed and protein was extracted using High-efficiency RIPA lysis buffer (Solarbio, China) after umbelliferone treatment (0, 5, 25, 50, 100 or 150 μmol L -1 ) as described above for 48 h. Equal amounts of protein were loaded onto SDS-PAGE before transfer onto PVDF membranes (0.2 μm, #162-0177 Bio-Rad, USA). After blocking with 5 % non-fat dry milk, the membranes were incubated with specific primary antibodies overnight at 4 °C. The following antibodies were used: anti-MCM2 (#12079), Cyclin E1 (#20808), Bax (#5023), Bcl-2 (#15071), phosphoinositide 3-kinase p110γ (#4252S), CDK2 (#2546S), CDK4 (#2906S), β-actin (#8457), GAPDH (#5174) (Cell Signaling Technology, USA, dilution 1:1,000), Cyclin D1 (PB0403) (Wuhan Boster Biological Technology, Ltd., China, dilution 1:1,000), and anti-Ki67 antibody (ab15580) (Abcam, UK, dilution 1:1,000). Secondary antibodies included goat anti-rabbit (BA1054) or goat anti-mouse (BA1050) IgG-HRP (Wuhan Boster Biological Technology, Ltd., China, dilution 1:5,000). Signals were detected by enhanced chemiluminescence (ECL) reagent (Beyotime). Densitometric analysis of proteins was performed using the Tanon GIS version 4.1.2 software (Tanon Science and Technology Co., Ltd., China).
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