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Rabbit anti ki67

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Rabbit anti-Ki67 is a primary antibody that binds to the Ki67 protein, a well-established marker of cell proliferation. It can be used in various immunodetection techniques, such as immunohistochemistry and immunofluorescence, to label proliferating cells.

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Close spelling variants of this product

Rabbit anti-mouse Ki67 Rabbit anti-Ki67 primary antibody Anti-Ki67 rabbit monoclonal antibody Rabbit anti-human Ki67 Rabbit anti-Ki67 antibody Rabbit anti-Ki67 antibody (ab16667) Rabbit anti-human Ki67 antibody Rabbit anti-Ki67 (ab15580) Rabbit monoclonal anti-Ki67 Rabbit polyclonal anti-Ki67

202 protocols using rabbit anti ki67

1

Ki67 Immunohistochemistry in Spheroids

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Spheroids were collected after treatment for 4 days and frozen in Optimum Cutting Temperature from Newcomer supply (Middleton, WI, USA). The spheroids were sectioned at 10 μm and incubated overnight with rabbit anti-Ki67 (Epitomics, Burlingame, CA, USA). Slides were incubated with biotinylated goat anti-rabbit (Dako, Campbellfield, Australia) and then with streptavidin (BD Pharmingen) before development with 3, 3′-diaminobenzidine tetrahydrochloride (DAB) (BD Pharmingen) and counterstaining with hematoxylin QS (Vector Laboratories).
Nehoff H., Parayath N.N., McConnell M.J., Taurin S, & Greish K. (2015). A combination of tyrosine kinase inhibitors, crizotinib and dasatinib for the treatment of glioblastoma multiforme. Oncotarget, 6(35), 37948-37964.
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2

Antibodies for Renal Protein Analysis

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The following primary antibodies were used: anti-AQP2 (7661AP) was kindly provided by Prof. Sebastian Frische; immunofluorescence (IF) and immunoblotting (IB) 1:1,000; rabbit anti-Ki67 (#4203–1, Epitomics, Cambridge, United Kingdom) IF 1:500; anti-B1/2 H+ATPase (#sc-55544, Santa Cruz, Dallas, TX, United States) IF and IB 1:1,000; rabbit anti-NKCC2 (#AB3562P, Millipore, Dramstad, Germany) IF and IB 1:1,000; rabbit anti-β1 integrin (#04-1109, Millipore, Dramstad Germany) IB 1:1,000; rabbit anti-ENaC alpha (SPC-403, StressMarq, Victoria, BC, Canada) IB 1:1000; mouse anti-β-Actin (#A2066 Sigma, Milan, Italy) IB 1:20,000; rabbit anti-GAPDH (#10018 GeneTex, Hsinchu City, Taiwan) IB 1:20,000. The following secondary antibodies were used: goat anti mouse Alexa Fluor 488 IF 1/800 (Invitrogen, CA, United States) goat anti-rabbit Cy3 (A10520, Invitrogen, CA, United States) IF 1:500; anti-mouse HRP conjugated (NA931V GE Healthcare, Little Chalfont, United Kingdom) IB 1:2,000; anti-rabbit HRP conjugated (NA934V GE Healthcare, Little Chalfont, United Kingdom) IB 1:2,000.
Iervolino A., De La Motte L.R., Petrillo F., Prosperi F., Alvino F.M., Schiano G., Perna A.F., Di Matteo D., De Felice M., Capasso G, & Trepiccione F. (2018). Integrin Beta 1 Is Crucial for Urinary Concentrating Ability and Renal Medulla Architecture in Adult Mice. Frontiers in Physiology, 9, 1273.
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3

Automated IHC Staining and Imaging

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Sections were stained using the Bond RX autostainer (Leica Biosystems Inc., Buffalo Grove, IL, USA). Briefly, slides were baked at 65 °C for 15 min and the automated system performed dewaxing, rehydration, antigen retrieval, blocking, primary antibody incubation, post primary antibody incubation, detection (DAB), and counterstaining using Bond reagents (Leica). Samples were then removed from the machine, dehydrated through a series of ethanol and xylenes and mounted. Rabbit anti-γ-H2AX (Ser139) (1:800, #9718, Cell Signaling), rabbit anti-phospho-EGFR (Tyr1068) (1:50, #2234, Cell Signaling), rabbit anti-phospho-HER2/ErbB2 (Tyr1221/22) (1:400, #2243, Cell Signaling), rabbit anti-PTEN (1:150, #9559, Cell Signaling: used for mouse transplants and normal reduction mammoplasty samples), mouse anti-PTEN (1:100, #M3627, Dako: used for HER2 breast cancer patient cohort), rabbit anti-Ki67 (1:200, #ab16667, Abcam) were diluted in antibody diluent (Leica). TUNEL staining was performed using manufacturer’s recommendations (ApopTag Peroxidase In Situ Apoptosis Detection Kit, #S7100; EMD Millipore). All microscopic imaging was done using the VECTRA® Automated Quantitative Pathology Imaging system or an Axioskop 40 with ZEN Software (Zeiss, Germany). Whole tissue imaging was done using a Stemi SV 11 Stereoscope with ZEN Software (Zeiss).
Sizemore G.M., Balakrishnan S., Thies K.A., Hammer A.M., Sizemore S.T., Trimboli A.J., Cuitiño M.C., Steck S.A., Tozbikian G., Kladney R.D., Shinde N., Das M., Park D., Majumder S., Krishnan S., Yu L., Fernandez S.A., Chakravarti A., Shields P.G., White J.R., Yee L.D., Rosol T.J., Ludwig T., Park M., Leone G, & Ostrowski M.C. (2018). Stromal PTEN determines mammary epithelial response to radiotherapy. Nature Communications, 9, 2783.
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4

Antibody-based Protein Expression Analysis

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Antibodies used were rabbit anti‐LAMP2A (# ab125068; Abcam), mouse anti‐β‐actin (# A5316; Sigma‐Aldrich), rabbit anti‐caspase‐3 (# 14220; Cell Signaling Technology), rabbit anti‐Ki67 (# ab15580; Abcam), rabbit anti‐p53 (# 9282; Cell Signaling Technology), rabbit anti‐Bax (# 2870; Cell Signaling Technology), rabbit anti‐Bcl‐2 (# ab32503; Abcam), and rabbit anti‐GAPDH (# 5174; Cell Signaling Technology). Cisplatin (CDDP) was obtained from Yakult Co., Ltd. Paclitaxel (PTX) was obtained from FUJIFILM Wako.
Ichikawa A., Fujita Y., Hosaka Y., Kadota T., Ito A., Yagishita S., Watanabe N., Fujimoto S., Kawamoto H., Saito N., Yoshida M., Hashimoto M., Minagawa S., Hara H., Motoi N., Yamamoto Y., Ochiya T., Araya J, & Kuwano K. (2020). Chaperone‐mediated autophagy receptor modulates tumor growth and chemoresistance in non–small cell lung cancer. Cancer Science, 111(11), 4154-4165.
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5

Immunohistochemical Analysis of CCL18 and Ki67

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Paraffin sections were deparaffinized with xylene for 10 min and rehydrated with graded alcohol. Antigen retrieval was performed by boiling the sections in 0.1 M citric acid buffer (pH 6.0) at 120°C for 10 min in a decloaking chamber (Biocare Medical, Walnut Creek, CA). After natural cooling, the sections were washed 3 times with PBS for 5 min each time. Then, the sections were stained with rabbit anti-CCL18 IgG Ab (20 μg/ml, Abcam) and rabbit anti-Ki67 (2 μg/ml, Abcam). Stayed overnight on a shaker at 4°C. After incubation, the slices were washed with PBS for 3 times, 5 min each time, then added hypersensitivity two-step immunohistochemical detection reagent, incubated at room temperature for 20 min; washed 3 times with PBS, 5 min each time, DAB (Sigma-Aldrich) was used for color development. Then hematoxylin complex dyeing, gradient alcohol dehydration, xylene transparent and sealed. The sections were observed, photographed, and counted under a light microscope.
The apoptosis of tumor was detected by TUNEL staining (Abcam), and the operation procedure was referred to the instruction manual.
Shang D., Liu Y, & Chen Z. (2022). Exosome-Transmitted miR-128 Targets CCL18 to Inhibit the Proliferation and Metastasis of Urothelial Carcinoma. Frontiers in Molecular Biosciences, 8, 760748.
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6

Immunofluorescence Staining of Macrophages

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Paraformaldehyde-fixed BMDM, peritoneal macrophages, and J774 from each group were washed with PBS and permeabilized with 0.1% Triton X-100/PBS at room temperature for 10 min. Samples were then blocked in 5% rabbit serum in PBS for 1 h at room temperature and incubated at 4 °C overnight with the following primary antibodies at 1:200 dilutions in 5% rabbit serum: rabbit anti-Ki67 (Abcam, Cambridge, UK), mouse-anti-iNOS (Abcam, Cambridge, UK), and rabbit anti-Arg-1 (Invitrogen, Waltham, MA, USA). Samples were washed three times for 5 min with PBS. They were then incubated for 1 h at room temperature with the following secondary antibodies at 1:400 dilutions in 5% rabbit serum: anti-rabbit Alexa Fluor 488 (Invitrogen), anti-mouse Alexa Fluor 594, and Alexa Fluor™ 594 Phalloidin at 1:40 dilutions (Invitrogen). Samples were then washed with PBS three times for 5 min each. Samples were then counter-stained with DAPI in Fluoroshield mounting medium (Sigma, St. Louis, MO, USA). The samples were analyzed by fluorescence microscopy (AXIO Imager D2, Zeiss) at a 20× magnification. A total depth of 20 µm was acquired for each sample and the total projection was visualized in the xy planes.
Wang C.S., Luo S.D., Jia S., Wu W., Chang S.F., Feng S.W., Yang C.H., Lin J.H, & Wee Y. (2022). Balance of Macrophage Activation by a Complex Coacervate-Based Adhesive Drug Carrier Facilitates Diabetic Wound Healing. Antioxidants, 11(12), 2351.
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7

Comprehensive Histological and Immunohistochemical Analysis of Mouse Tissues

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Images of the mice and their organs were obtained using a digital fluorescent microscope (VB-6000; Keyence Japan, Osaka, Japan, or Leica AF6500; Leica Microsystems, Tokyo, Japan). For histological analyses, tissues were fixed in 10% formalin solution and embedded in paraffin for hematoxylin and eosin (H&E) staining. Alternatively, tissues were fixed by perfusion with 4% paraformaldehyde. Frozen sections were the cut using a Microm HM500 OM Microtome Cryostat (Carl Zeiss Japan, Tokyo, Japan).
The following primary antibodies were used for immunohistochemical analysis: guinea pig anti-insulin, rabbit anti-glucagon, rabbit anti-somatostatin, goat anti-pancreatic polypeptide, rabbit anti-chromograninA (cgA), rabbit anti-Ki67, and rabbit anti-VEGF (Abcam, Tokyo, Japan). Alexa568- or Cy3- labeled species-specific anti-IgG antibodies (Life Technologies Japan, Tokyo, Japan) were used as secondary antibodies. Images were obtained using a LSM710 laser scanning microscope (Carl Zeiss Japan), or a HS BZ-9000 fluorescent microscope system (Keyence). The Ki-67 index was calculated by dividing the total number of nuclei by the number of Ki-67-positive nuclei. The number of nuclei was counted by observing 8–10 fields with a 40× lens.
Takano Y., Kasai K., Takagishi Y., Kikumori T., Imai T., Murata Y, & Hayashi Y. (2015). Pancreatic Neuroendocrine Tumors in Mice Deficient in Proglucagon-Derived Peptides. PLoS ONE, 10(7), e0133812.
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8

Protein Extraction and Western Blot Analysis

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Pieces of whole liver were homogenized using a tissue homogenizer (TissueRuptor, Qiagen, Germantown, MD), followed by centrifugation. Protein extraction from cells or whole liver was performed using lysis buffer complemented with protease inhibitor (Complete Lysis M kit, Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitor (Halt Phosphatase Inhibitor Single-Use Cocktail, Thermo Scientific, Waltham, MA). Protein concentration was measured using a Bio-Rad DC kit (Bio-Rad Laboratories, Hercules, CA) and lysates were then subjected to immunoblot analysis. Blots were developed with the ECL Detection System (Amersham, Pharmacia Biotech, Buckinghamshire, England). Densitometry of bands was performed with ImageJ software (rsbweb.nih.gov/ij, NIH, Bethesda, MD). Western blot membranes were incubated with the following primary antibodies: Rabbit anti-β-PDGFR (1:500) and anti-phospho-β-PDGFR (1:500) were obtained from Santa Cruz Biotechnology, Santa Cruz, CA, rabbit anti-CD31 (1:500), rabbit anti-Ki67 (1:2500), rabbit anti-Desmin (1:1000) and rabbit anti-Calnexin (1:3,000) were from Abcam, Cambridge, England). The reactions were detected with the Fujifilm LAS-4000 system (Fujifilm Life Science, Stamford, CT), using a horseradish peroxidase-conjugated secondary antibody (Anti-rabbit IgG, Cell Signaling, Danvers, MA).
Kocabayoglu P., Zhang D.Y., Kojima K., Hoshida Y, & Friedman S.L. (2015). Induction and Contribution of β-PDGFR Signaling by Hepatic Stellate Cells to Liver Regeneration after Partial Hepatectomy in Mice. Liver international : official journal of the International Association for the Study of the Liver, 36(6), 874-882.
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9

Multimarker IHC Analysis of Signaling Pathways

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Immunohistochemistry (IHC) staining for Shh, c-Met, E-Cadherin, IGF-1 and Ki67 were performed by hand. Tissue blocks with poor quality were excluded from the study. The slides for all stainings were hydrated; antigen retrieval was performed in a pressure cooker with citrate buffer (pH 6.0) for Shh and Ki67, in a steamer with citrate buffer (pH 6.0) for c-Met and IGF-1 and with Tris-EDTA buffer (pH 9.0) for E-Cadherin. Then, the slides were blocked in peroxidase, avidin and biotin block sequentially. Goat-anti-Shh (R&D), rabbit-anti-c-Met (Santa Cruz), rabbit anti-IGF-1 (Abcam), rabbit anti-E-Cadherin (Abcam), or rabbit anti-Ki67 (Abcam) primary antibodies at 1:50 (Shh, c-Met, E-Cadherin), 1: 500 (Ki67) and 4µg/ml (IGF-1) were added, and the slides were incubated for 1 hour at room temperature. Then, rabbit anti-goat or goat anti-rabbit biotinylated secondary antibodies (Vector Laboratories) respectively, were added for 30 minutes at room temperature. The signal was amplified and detected using the ABC Vectastain kit (Vector Laboratories) according to the manufacturer’s instructions. The slides were developed using DAB and counterstained by hematoxylin. Lastly, the slides were dehydrated and mounted. All IHC slides were analyzed and scored by a pathologist. Immunofluorescence staining of c-Met and Shh was described in Supplementary Materials and Methods.
Rucki A.A., Xiao Q., Muth S., Chen J., Che X., Kleponis J., Sharma R., Anders R.A., Jaffee E.M, & Zheng L. (2017). Dual inhibition of Hedgehog and c-Met pathways for pancreatic cancer treatment. Molecular cancer therapeutics, 16(11), 2399-2409.
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10

Immunohistochemistry Protocol for Brain Sections

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The sections were processed under the same conditions to obtain comparable immunohistochemistry among groups as described in a previous study (Jung et al., 2019). Three sections from 1.82 to 2.32 mm posterior to the bregma according to a mouse atlas (Paxinos & Franklin, 2001), separated by intervals of 150 μm, were obtained from each animal. Each tissue section was sequentially treated with 0.3% H2O2 in PBS for 30 min and 10% normal goat serum in 0.1 M PBS for 30 min at 25°C. The sections were first incubated overnight with rabbit anti‐Ki67 (1:1,000; Abcam), rabbit anti‐doublecortin (DCX, 1:2,000, Abcam), rabbit anti‐glucose transporter 3 (GLUT3; 1:50, Santa Cruz Laboratory), or rabbit anti‐phosphorylated cAMP response element‐binding protein at Ser133 (pCREB, 1:400; Cell Signaling) at 25°C. The next day, the sections were treated with biotinylated goat anti‐rabbit IgG (1:200; Vector) for 2 hr at 25°C. Subsequently, the sections were treated with streptavidin–peroxidase complex (1:200; Vector) for 2 hr at 25°C. Thereafter, the brain sections were visualized by reaction with 3,3′‐diaminobenzidine tetrahydrochloride (DAB, Sigma) in 0.1 M Tris‐HCl buffer (pH 7.2) and mounted on gelatin‐coated slides. Sections were dehydrated with graded concentrations of alcohol and mounted in Canada balsam (Kanto Chemical).
Kim W., Hahn K.R., Jung H.Y., Kwon H.J., Nam S.M., Kim J.W., Park J.H., Yoo D.Y., Kim D.W., Won M., Yoon Y.S, & Hwang I.K. (2019). Melatonin ameliorates cuprizone‐induced reduction of hippocampal neurogenesis, brain‐derived neurotrophic factor, and phosphorylation of cyclic AMP response element‐binding protein in the mouse dentate gyrus. Brain and Behavior, 9(9), e01388.
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