Overview

  • Product nameAnti-Ki67 antibody
    See all Ki67 primary antibodies
  • Description
    Rabbit polyclonal to Ki67
  • Tested applicationsIHC - Wholemount, IHC-P, IHC-FrFl, Flow Cyt, IHC-Fr, ICC/IF, ICC, WB, IHC-FoFrmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Horse, Cow, Dog, Human, Pig, Indian Muntjac, Monkey, Chinese Hamster, Marmoset (common), Syrian Hamster
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1200 - 1300 of Human Ki67.

    (Peptide available as ab15581.)

  • Positive control
    • WB: Hela whole cell lysate IF: mouse (P0) olfactory bulb, MEF1 IHC-Fr: mouse P7 brain sections IHC-P: mouse spleen

Properties

Applications

Our Abpromise guarantee covers the use of ab15580 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC - Wholemount Use at an assay dependent concentration.
IHC-P Use a concentration of 0.1 - 10 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
IHC-FrFl 1/500. (see Abreview)
Flow Cyt 1/100.
IHC-Fr 1/100 - 1/1000.
ICC/IF 1/100 - 1/1000.
ICC Use at an assay dependent dilution.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 345, 395 kDa (predicted molecular weight: 359 kDa).Can be blocked with Ki67 peptide (ab15581).
IHC-FoFr Use at an assay dependent dilution.

Target

Anti-Ki67 antibody images

  • All lanes : Anti-Ki67 antibody (ab15580) at 1 µg/ml

    Lane 1 : Hela whole cell lysate
    Lane 2 : Hela whole cell lysate with Ki67 peptide (ab15581) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    Alexa Fluor Goat polyclonal to Rabbit IgG at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size : 359 kDa
  • Fluorescent confocal microscopy (20x) of mouse (P0) olfactory bulb, outer glomeruli layer, showing Ki67 immunoreactivity (ab15580; 1/1000; overnight at RT, 0.25% TX-100 no blocking step) using a secondary goat anti-rabbit fluorescent antibody (Alexa Fluor 488;1/300 2h at RT.

  • ab15580 staining Ki67 in SK-N-SH cells treated with NADA (N-Arachidonyldopamine) (ab120099), by ICC/IF. Decrease in Ki67 expression correlates with increased concentration of NADA (N-Arachidonyldopamine), as described in literature.
    The cells were incubated at 37°C for 10 minutes in media containing different concentrations of ab120099 (NADA (N-Arachidonyldopamine)) in ethanol, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab15580 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.
  • SK-N-SH cells were permitted to grow to confluency, then serum starved for 48 hours and predominantly driven into G0. The cells were then paraformaldehyde fixed and immunofluorescently labelled with anti-Ki67 (ab15580) at a dilution of 1/1000. The majority of the cells show little or no Ki67 staining, indicating they are in G0 arrest (red cells). Two cells however show strong nucleolar Ki67 staining indicating they are still cycling (green cells). The DNA is stained with DAPI and is shown in red. The Ki67 staining is shown in green. x 63 magnification.

    Similar results were seen with an asynchronous population of HeLa cells. The Ki67 staining was localised to the periphery of the nucleoli and throughout the nucleoplasm of proliferating cells. (This data is not shown but is available upon request).

  • Immunostaining for Rabbit polyclonal to Ki67 - Proliferation Marker (ab15580) on Mouse P7 brain (frozen) sections. Sections were fixed with paraformaldehyde, neither a blocking nor antigen retrieval steps were included in this protocol. Secondary antibody used was Rabbit IgG antibody (ab7082; 1/250).
  • ab15580 at 1/50 staining Human umbilical artery endothelial cells by ICC/IF. The tissue was paraformaldehyde fixed and blocked before permeabilization with saponin and incubation with the antibody for 16 hours. A FITC conjugated goat anti-rabbit IgG was used as the secondary. The merged image shows those cells expressing Ki67 from the total number of exponential cells.

    See Abreview

  • IHC image of ab15580 stained human skin carcinoma FFPE section. Section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30 seconds at 125°C. Section was incubated with ab15580 at a dilution of 1:200 for 1h at room temperature and detected using an HRP conjugated polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  • ab15580 staining Ki67-Proliferation Maker in human colon tissue sections by IHC-P (formaldehyde-fixed paraffin-embedded sections). Tissue samples were fixed with formaldehyde and blocked with 10% goat serum  for 30 minutes at 25°C. Antigen retrieval was by heat mediation in Target Retrieval Solution. Samples were incubated with primary antibody 1/5000 (TBST) for 1 hour at 25°C. 

    See Abreview

  • ICC/IF image of ab15580 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab15580, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • ICC/IF image of ab15580 stained MEF1 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab15580, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
  • IHC image of ab15580 staining in mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol B. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab15580, 1µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  • ab15580 staining Ki67 - Proliferation Marker in Human PBMCs by Flow Cytometry. Cells were isolated by Ficoll density separation, fixed in paraformaldehyde and permeabilized in 0.1% Saponin + PBS. The sample was incubated with the primary antibody (1/100 in PBS + 1% FCS and 0.09% sodium azide) for 1 hour at 4°C. A phycoerythrin-conjugated Goat anti-rabbit IgG (1/100) was used as the secondary antibody.

    Gating Strategy: Lymphocytes and Monocytes

    1 = isotype; 2 = PBMCs untreated; 3 = PBMCs treated with PHA.

    See Abreview

  • ab15580 staining Ki67 - Proliferation Marker in Human skin tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 4% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in a citrate buffer (pH 6.0). Samples were incubated with primary antibody (5 µg/ml in blocking buffer) for 16 hours at 4°C. A Texas Red ® Goat anti-rabbit IgG polyclonal (1/100) was used as the secondary antibody.

    See Abreview

References for Anti-Ki67 antibody (ab15580)

This product has been referenced in:
  • Magri L  et al. E2F1 coregulates cell cycle genes and chromatin components during the transition of oligodendrocyte progenitors from proliferation to differentiation. J Neurosci 34:1481-93 (2014). Read more (PubMed: 24453336) »
  • Postnikov YV  et al. Loss of the nucleosome-binding protein HMGN1 affects the rate of N-nitrosodiethylamine-induced hepatocarcinogenesis in mice. Mol Cancer Res 12:82-90 (2014). IHC ; Mouse . Read more (PubMed: 24296759) »

See all 173 Publications for this product

Product Wall

Our lots of ab15580 that have a concentration less than 1 mg/mL have BSA added. Thus, since your lot has a concentration of 0.6 mg/mL, BSA is included in your vial of ab15580.

Application Immunocytochemistry
Blocking step FBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Sample Human Cultured Cells (Immortalized myoblasts)
Specification Immortalized myoblasts
Permeabilization No
Fixative Paraformaldehyde
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Submitted Sep 02 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 22°C
Sample Human Cell (Keratinocyte)
Specification Keratinocyte
Permeabilization Yes - 0.1% Triton X-100 in PBS
Fixative Formaldehyde
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Submitted Sep 02 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step FCS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Sodium Citrate ph6.0 Decloaking Chamber 125 degrees 5 minutes
Sample Human Tissue sections (KHOS cells in Balbc nu/nu mouse)
Specification KHOS cells in Balbc nu/nu mouse
Permeabilization Yes - 0.1% Triton X-100 in TBS
Fixative Formaldehyde
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Submitted Sep 02 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Tris better than Cit
Sample Human Tissue sections (Skeletal Muscle)
Specification Skeletal Muscle
Permeabilization No
Fixative Methacarn
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Submitted Jun 25 2014

Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (intestine)
Specification intestine
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 5%
Fixative Paraformaldehyde
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Submitted Jun 11 2014

Application Immunohistochemistry (Frozen sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Sample Mouse Tissue sections (Postnatal day 3 testes)
Specification Postnatal day 3 testes
Permeabilization No
Fixative 4%PFA
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Dr. Qing Wen

Verified customer

Submitted May 05 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 18°C
Sample Human Cell (hippocampal progenitor cells)
Specification hippocampal progenitor cells
Permeabilization Yes - Triton X-100
Fixative Paraformaldehyde
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Dr. Aleksandra Maruszak

Verified customer

Submitted Apr 28 2014

Application Immunohistochemistry (Frozen sections)
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Sample Mouse Tissue sections (spinal cord (post spinal cord injury), 25 micron)
Specification spinal cord (post spinal cord injury), 25 micron
Permeabilization Yes - Triton X-100
Fixative Formaldehyde
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Submitted Apr 23 2014

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: pH6 Citrate, pressure cooker
Sample Mouse Tissue sections (Human derived tumour in mouse lung)
Specification Human derived tumour in mouse lung
Permeabilization No
Fixative Formaldehyde
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Submitted Mar 12 2014

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