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Synthetic peptide conjugated to KLH derived from within residues 1200 - 1300 of Human Ki67.
(Peptide available as ab15581.)
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.
|IHC - Wholemount||Use at an assay dependent concentration.|
|IHC-P||Use a concentration of 0.1 - 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|IHC-FrFl||1/500. (see Abreview)|
ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.
|ICC/IF||Use a concentration of 0.5 - 1 µg/ml.|
|ICC||Use at an assay dependent concentration.|
ab15580 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab15580 at 1μg/ml concentration and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat anti-rabbit IgG Alexa Fluor® 488 (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
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) for 20 mins. The section was then incubated with ab15580, 5µg/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.
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 donkey anti-rabbit IgG biotin secondary antibody (ab7082; 1/250).
IHC image of Ki67 staining in human spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) for 20 mins. The section was then incubated with ab15580, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chrogen. 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.
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 goat anti-rabbit DyLight 488 secondary 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).
Immunohistochemistry analysis of Formaldehyde-fixed paraffin-embedded mouse tumour tissue sections labelling Ki67 with ab15580 at 1/2000. The secondary antibody was biotin conjugated goat polyclonal vector at a dilution of 1/250.
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, goat anti-rabbit IgG DyLight® 488 secondary antibody 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, goat anti-rabbit DyLight® 488 (H+L) secondary antibody 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.
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