IHC image of ab16667 staining Ki67 in a section of formalin-fixed paraffin-embeddedrat spleen tissue. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0).. The section was then incubated with ab16667, 1/200 dilution and detected using Goat Anti-Rabbit IgG H&L (HRP) antibody. DAB was used as the chromogen. The section was then counterstained with haematoxylin.

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.

IHC image of ab16667 staining Ki67 in a section of formalin-fixed paraffin-embedded normal human tonsil tissue. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0).. The section was then incubated with ab16667, 1/200 dilution and detected using Goat Anti-Rabbit IgG H&L (HRP) antibody. 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.

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 sodium citrate buffer (pH 6.0).. The section was then incubated with ab16667, 1/200 dilution and detected using Goat Anti-Rabbit IgG H&L (HRP) antibody. DAB was used as the chromogen. The section was then counterstained with haematoxylin.

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 withGoat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibodyat 2μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

This image was generated from the hybridoma version.

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).

Effect of newborn anoxia and PD156707 on proliferation and binucleation of neonatal cardiomyocytes

A representative image of cardiomyocytes stained with α-actinin (green), Ki-67 (red), and Hoescht (blue).

4% paraformaldehyde-fixed, Triton X-100 permeabilized rat cardiomyocytes were stained for Ki67 using ab16667 at 1/100 dilution in ICC/IF.

(From Figure 3A of Paradis et al)

This image was generated from the hybridoma version.

Immunocytochemistry/ Immunofluorescence analysis of human cardiac stem cells labelingKi67 withab16667 at 1/250 dilution. Cells were fixed in paraformaldehyde and permeabilized withTriton 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 for2 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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