Anti-Ki67 antibody - Proliferation Marker (ab15580)

Western blot - Ki67 antibody (ab15580)

Observed band sizes : 345kDa, 395kDa

Immunofluorescence - Ki67 antibody (ab15580)

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.

Image courtesy of Julien Laffaire, Laboratoire de Neurobiologie, ESPCI, Paris, France

Immunofluorescence - Ki67 antibody - Proliferation Marker (ab15580)

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).

Kirk McMannus, University of British Columbia

Immunohistochemistry (Frozen sections) - Ki67 antibody - Proliferation Marker (ab15580)

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).

Randal Moldrich, CNRS UMR7637, ESPCI, France

Immunocytochemistry/ Immunofluorescence - Ki67 antibody - Proliferation Marker (ab15580)

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.

This image is courtesy of an Abreview submitted by Dr Jose Javier Martin De Llano

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Ki67 antibody - Proliferation Marker (ab15580)

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.

Immunohistochemistry (Frozen sections) - Ki67 antibody - Proliferation Marker (ab15580)

Ki67 expression in the sub-ventricular zone of a 1 month-old mouse brain

Ki67 staining using ab27619 (1/500) in the sub-ventricular zone of a 1 month-old mouse brain. The mouse has been perfused with paraformaldehyde 4% (50ml). After dissection, the brain has been incubated overnight in sucrose 20%, embedded in OCT and cryosectioned (10 µm). Sections have been treated for antigen retrieval (20 minutes at 60C). The secondary antibody used was a non-Abcam Goat anti-rabbit AlexA568.

Francois Guillemot, NIMR, UK

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Ki67 antibody - Proliferation Marker (ab15580)

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. 

This image is courtesy of an anonymous Abreview

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody - Proliferation Marker (ab15580)

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.

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody - Proliferation Marker (ab15580)

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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody - Proliferation Marker (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, 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.