Anti-Ki67 抗体 (ab15580)

IHC image of ab15580 staining in mouse spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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.

Paraformaldehyde-fixed Rabbit cell (Retina) labeling Ki67 (Green) using ab15580 at 1/200 dilution followed by a Donkey anti-rabbit Alexa Fluor® 568 secondary antibody in ICC/IF analysis. Normal Donkey serum was used as the blocking agent for 15 hours at 4ºC. 

Tissue was immersion fixed in 4% paraformaldehyde overnight at 4 degrees Celsius. Tissue was then embedded in 10% agarose and section at 100 microns. Sections were placed in 2N HCL for 1 hour before commencing immunocytochemistry. Ki-67 (dividing cells red).

IHC image of Ki67 staining in human spleen formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM 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 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 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.

Confocal images of mouse trachea epithelium collected at steady state, 24 and 48 h after SO₂ injury. Tissue sections were co-stained with UHRF1 and Ki67, a proliferation marker. 

ab15580 was used to stain Ki67 at a dilution of 1:1 000

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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.

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.

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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).

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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.  

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ab15580 staining Ki67 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), 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 overnight at +4°C with ab15580 at 1ug/ml then detected with an Alexa Fluor^® 488 goat anti-rabbit secondary antibody (ab150081) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

ab15580 staining Ki67 in MEF1 cells. The cells were fixed with 4% paraformaldehyde (10 min), 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-Triton for 1h. The cells were then incubated with the antibody ab15580 at 1µg/ml and ab7291 (Mouse monoclonal to alpha Tubulin - Loading Control) used at a 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed, (pseudo-colored green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor® 594) preadsorbed, (colored red), both used at a 1/1000 dilution for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature.