All lanes : Anti-Ki67 antibody [SP6] (ab16667) at 1/100 dilution

Lane 1 : Ramos cell lysate
Lane 2 : Wild-type HeLa cell lysate
Lane 3 : MKI67 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 358 kDa
Observed band size: 359 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab16667).

Lanes 1-3: Merged signal (red and green). Green - ab16667 observed at 359 kDa. Red - loading control, ab130007 observed at 125 kDa.

ab16667 was shown to react with Ki67 in wild-type HeLa. Loss of signal was observed when knockout sample ab263762 was used. Wild-type and Ki67 knockout samples were subjected to SDS-PAGE. ab16667 and Anti-Vinculin antibody VIN-54] (ab130007) were incubated overnight at 4°C at 1 in 100 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773)  and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation (ab16667).

 

Fluorescence multiplex immunohistochemical analysis of normal human tonsil tissue (formalin-fixed paraffin-embedded section).

Merged staining of anti-PD1 (ab237728; orange; Opal™520), anti-PDL1 (ab237726; green; Opal™540), anti-CD68 (ab192847; yellow; Opal™570), anti-CD3 (ab16669; red; Opal™620), anti-Ki67 (ab16667; light blue; Opal™650) and anti-PanCK (ab7753; grey; Opal™690).

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument with an Opal™ 7-color automation IHC kit (NEL821001KT, Akoya Biosciences^®).

The section was incubated in six rounds of staining; in the order of ab237728 (1/500 dilution), ab237726 (1/500 dilution), ab192847 (1/300 dilution), ab16669 (1/300 dilution), ab16667 (1/200 dilution) and ab7753 (1/200 dilution); each using a separate fluorescent tyramide signal amplification system.

Sodium citrate antigen retrieval (Leica ER1, pH6.0, 30 minutes) was used in between rounds of tyramide signal amplification to remove the antibody from the previous round, to avoid any cross-reactivity.

DAPI (dark blue) was used as a nuclear counter stain.

Microscopy and pseudocoloring of individual Opal™ dyes was performed using a Vectra Polaris.

This image was generated from the hybridoma version.

This data was developed using the same antibody clone in a different buffer formulation (ab16667).

 

ab16667 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol for 5 minutes, 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 1 hour. The cells were then incubated with ab16667 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

This image was generated from the hybridoma version.

This data was developed using ab16667, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed, None permeabilized parental HAP1 cells labelling Ki67 with ab16667 at 1/1000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing nucleolar staining in parental HAP1cell line ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1000 dilution (2 µg/mL).

This data was developed using ab16667, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed, None permeabilized HeLa cells labelling Ki67 with ab16667 at 1/1000 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (2 µg/mL) (Green). Confocal image showing nucleolar staining in HeLa cell line ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1000 dilution (2 µg/mL).

This data was developed using the same antibody clone in a different buffer formulation (ab16667).

 

IHC image of ab16667 staining Ki67 in a section of formalin-fixed paraffin-embedded normal human tonsil* 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 (pH 6.0, epitope retrieval solution 1) for 20mins. The section was then incubated with ab16667, 1/200 dilution, 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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

This image was generated from the hybridoma version.

This data was developed using the same antibody clone in a different buffer formulation (ab16667).

 

ab16667 staining Ki67 in rat oesophagus by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer. Samples were then blocked with 1% BSA for 10 minutes at 21ºC followed by incubation with the primary antibody for 30 minutes at 1/100. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/250 dilution.

This image was generated from the hybridoma version.

This data was developed using ab16667, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized parental HAP1 (Wildtype control Human chronic myelogenous leukemia near-haploid cell line) cells labelling Ki67 with ab16667 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.