Anti-Ki67 抗体 [SP6] (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.

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.

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.

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.

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Immunostained tissue sections from control-, AT1-, and MLL-LNs

Proliferating cells (Ki67+, brown) were seen in lymph follicles (presumably B-lymphocytes) and in para-follicular areas (presumably T-lymphocytes) in control-LNs. In AT1-LNs, the para-follicular areas dominated, and contained most of the proliferating cells. In MLL-LNs, the para-follicular regions appeared to contain less proliferating cells than in AT1-LNs.

Frozen sections of rat lymph node tissue were stained for Ki67 using ab16667 in immunohistochemical analysis. DAB staining.

(From Figure 10A of Strömvall et al)

This image was generated from the hybridoma version.

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.

ab16667 staining Ki67 in common marmoset spleen 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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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. 

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ab16667 staining Ki67 in rat liver tissue sections by immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 1% BSA for 2 hours at 22°C. Samples were incubated with primary antibody (1/500 in PBS-T + 1% BSA) for 18 hours at 4°C. A biotin-conjugated goat anti-rabbit IgG monoclonal (1/2000) was used as the secondary antibody.

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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 in mouse embryonic skin tissue 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 16 hours at 1/50. 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 in rat small intestine tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using 10 mM citrate buffer pH 6.0. Samples were then blocked with 10% serum for 20 minutes at room temperature followed by incubation with the undiluted primary antibody for 30 minutes. A biotin-conjugated goat anti-rabbit polyclonal was used as secondary antibody at a 1/2000 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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