Recombinant Anti-Ki67 antibody [37C7-12] (ab245113)

ab245113 staining Ki67 in Hap1-Ki67 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-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 ab245113 at 1µg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 4% paraformaldehyde (10 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling Ki67 with ab245113 at 1/800 dilution, followed by a ready to use secondary. Nuclear staining on human breast carcinoma tissue is observed. The section was incubated with ab245113 for 30 mins at RT. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, followed by ready to use secondary. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Ki67 with ab245113 at 1/100 dilution, followed by Goat Anti-Mouse IgG (Alexa Fluor^® 488) (ab150113) secondary antibody at 1/1000 dilution (green). Confocal image showing nucleolus staining on HeLa cell line. The nuclear counterstain is DAPI (blue).
Tubulin is detected with ab179504 Anti-beta IV Tubulin antibody - Microtubule Marker  at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor®  594) (ab150080) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab245113 at 1/50 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/1000 dilution.
-ve control 2: ab179504 at 1/200 dilution, followed by ab150113 AlexaFluor^®488 Goat anti-Mouse secondary at 1/1000 dilution.

Intracellular Intracellular Flow Cytometry overlay histogram showing wild-type HAP1 (green line) and MKI67 knockout HAP1 cells stained with ab245113 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab245113) (1x10⁶ in 100 µl at 0.2 µg/ml) for 30 min at 22°C. 

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150117) was used at 1/2000 for 30 min at 22°C. 

Isotype control antibody was mouse IgG2a&kappa (ab18413) used at the same concentration and conditions as the primary antibody (wild-type HAP1 - black line MKI67 knockout HAP1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). 

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. This antibody can also be used in HAP1 cells fixed with 80% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions. 

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Ki67 with ab245113 at 1/800 dilution, followed by a ready to use secondary. Nuclear staining on human tonsil tissue is observed. The section was incubated with ab245113 for 30 mins at RT. The immunostaining staining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, followed by ready to use secondary. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HAP1 (human chronic myelogenous leukemia near-haploid cell line) cells labeling Ki67 with ab245113 at 1/100 dilution, followed by Goat Anti-Mouse IgG (Alexa Fluor^® 488) (ab150113) secondary antibody at 1/1000 dilution (green). Confocal image showing nucleolus staining on HAP1 cell line. The nuclear counterstain is DAPI (blue).
Tubulin is detected with ab179504 Anti-beta IV Tubulin antibody - Microtubule Marker  at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab245113 at 1/50 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 594) (ab150080) secondary antibody at 1/1000 dilution.
-ve control 2: ab179504 at 1/200 dilution, followed by ab150113 AlexaFluor^®488 Goat anti-Mouse secondary at 1/1000 dilution.