Anti-Ki67 antibody [SP6] - BSA and Azide free

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

This data was developed using ab16667, the same antibody clone in a different buffer formulation. Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil labelling PD1 with ab243644 at 1/500 dilution (1.02 µg/mL) (D), Ki67 with ab16667 at 1/200 dilution (0.15 μg/ml) (B) and PD-L1 with ab228462 at 1/100 dilution (0.52 μg/ml) (C). Opal Polymer HRP Ms + Rb was used as a secondary antibody, and DAPI was used for a nuclear counter stain. Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins. Panel A : merged staining of anti-Ki67 (magenta; Opal™690), anti-PD-L1 (red; Opal™570) and anti-PD1 (green; Opal™520) on human tonsil. Panel B : anti-Ki67 stained on nucleus of proliferating cells. Panel C : anti-PD-L1 stained on membrane of cells involved in T cell inhibition. Panel D : anti-PD1 stained on antigen-stimulated T cells. The section was incubated in three rounds of staining : in the order of ab16667 for 10 mins, ab243644 for 30 mins and ab228462 for 10 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil Ki67 with ab197547 at a concentration of 5µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab197547 anti Ki67 antibody was incubated at 37oC for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling Ki67 with ab16667 at 1/500 dilution. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32min with ULTRA cell conditioning solution (CC1 pH8.5). ab16667 anti Ki67 antibody was incubated at 37°C for 16min. Sections were counterstained is with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized parental HAP1 (Wildtype controlHuman chronic myelogenous leukemianear-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.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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 Human tonsil tissue. The section was pre-treated using heat mediated antigen retrieval with ab93678 (citrate buffer, pH 6.0). The section was then incubated with ab16667, 1/200 (0.156 μg/ml) dilution and detected using ready to use Goat Anti-Rabbit IgG H&L (HRP) antibody. Nuclear staining on human tonsil is observed. The section was incubated with ab16667 overnight at +4°C. DAB was used as the chromogen. The section was then counterstained with haematoxylin.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

This data was developed using the same antibody clone in a different buffer formulation (ab16667).
Immunohistochemical analysis of formalin-fixed paraffin-embedded human tonsil sections labeling Ki67 with ab16667 at 1/200 dilution (0.156 μg/mL).

Image A :
Secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer)
Human tonsil tissue incubated with ab16667 overnight at +4°C.
Heat mediated antigen retrieval using ab93678 (citrate buffer pH 6.0).
Nuclear staining on human tonsil. The section was incubated with ab16667 overnight at +4°C.

Image B :
Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection)
Human tonsil tissue incubated with ab16667 on a Leica Biosystems BOND^® RX instrument.
Heat mediated antigen retrieval with Citrate buffer (pH 6.0 epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

Tissue Microarrays stained for "Anti-Ki67 antibody [SP6]” using "ab16667" at 1/200 dilution (0.145 μg/ml) in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negative (cross mark) staining per sample type tested. The sections were pre-treated using Heat mediated antigen retrieval using ab93678 (citrate buffer, pH 6.0). The sections were incubated with ab16667 at +4°C overnight followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880). Hematoxylin was used as the counter stain. This data was developed using the same antibody clone in a different buffer formulation (ab16667).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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 Rat spleen tissue. The section was pre-treated using heat mediated antigen retrieval with (citrate buffer, pH 6.0). The section was then incubated with ab16667, 1/200 (0.156 μg/mL) dilution and detected using ready to use Goat Anti-Rabbit IgG H&L (HRP) antibody. Nuclear staining on rat spleen. The section was incubated with ab16667 overnight at +4°C. DAB was used as the chromogen. The section was then counterstained with haematoxylin.

• IHC-P

Collaborator

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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 21°C 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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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 Mouse spleen tissue. The section was pre-treated using heat mediated antigen retrieval with ab93678 (citrate buffer, pH 6.0). The section was then incubated with ab16667, 1/200 (0.156 μg/mL) dilution and detected using a ready to use Goat Anti-Rabbit IgG H&L (HRP) antibody. Nuclear staining on mouse spleen.The section was incubated with ab16667 overnight at +4°C. DAB was used as the chromogen. The section was then counterstained with haematoxylin.

• WB

Lab

Western blot - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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.

All lanes:

Western blot - Anti-Ki67 antibody [SP6] (<a href='/en-us/products/primary-antibodies/ki67-antibody-sp6-ab16667'>ab16667</a>) at 1/100 dilution

Lane 1:

Ramos cell lysate at 20 µg

Lane 2:

Wild-type HeLa cell lysate at 20 µg

Lane 3:

Western blot - Human MKI67 (Ki67) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-mki67-ki67-knockout-hela-cell-line-ab255407'>ab255407</a>) at 20 µg

Lane 3:

Western blot - Human MKI67 (Ki67) knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-mki67-ki67-knockout-hela-cell-lysate-ab263762'>ab263762</a>) at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 358 kDa

Observed band size: 124 kDa,359 kDa

false

• WB

Lab

Western blot - Anti-Ki67 antibody [SP6] - BSA and Azide free (AB197547)

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

Western blot : Anti-MKI67 antibody [SP6] (ab16667) staining at 1/500 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab16667 was shown to bind specifically to MKI67. A band was observed at 359 kDa in wild-type A549 cell lysates with no signal observed at this size in MKI67 knockout cell line. To generate this image, wild-type and MKI67 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Ki67 antibody [SP6] (<a href='/en-us/products/primary-antibodies/ki67-antibody-sp6-ab16667'>ab16667</a>) at 1/500 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

MKI67 knockout A549 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 359 kDa

false