Recombinant Anti-Ki67 antibody [SP6] (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.

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 cells. Loss of signal was observed when knockout cell line ab255407 (knockout cell lysate 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.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded human tonsil sections labeling Ki67 with ab16667 at 1/200 (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 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 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. 

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.

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 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. 

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

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

Intracellular 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.

Overlay histogram showing HAP1 wildtype (green line) and HAP1-MKI67 knockout cells (red line) stained with ab16667. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab16667,1/1000) for 30 min at 22°C. The secondary antibody used wasGoat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed (ab150081) secondary antibodyat 1/2000 dilution for 30 min at 22°C.  A Rabbit IgG isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-MKI67 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).  Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.   This image was generated from the hybridoma version. 

Immunocytochemistry/ Immunofluorescence analysis of human cardiac stem cells labeling Ki67 with ab16667 at 1/250 dilution. Cells were fixed in paraformaldehyde and permeabilized with Triton x-100, 0.01%. Cells were blocked in BSA for 1 hour at room temperature. A polyclonal chicken anti-rabbit Alex Fluor^® 488 secondary antibody was used at 1/500 dilution. 

This image was generated from the hybridoma version.

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ab16667 staining Ki67 in human testis 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 2 hours 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.

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ab16667 staining Ki67 - Proliferation Marker in human HEp-2 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and permeabilized with Triton X-100 0.25% in PBS. Samples were incubated with primary antibody (1/50 in DPBS) for 1 hour at 21°C. An Atto488-conjugated Donkey anti-rabbit polyclonal (1/50) was used as the secondary antibody.

This image was generated from the hybridoma version.

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