Anti-Ki67 antibody [EPR3610] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

Flow cytometry overlay histogram showing wild-type Hap1 (green line) and MKI67 knockout Hap1 stained with ab92742 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab92742) (1x 106 in 100μl at 0.04 μg/ml (1/57000)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control was used at the same concentration and conditions as the primary antibody (wild-type Hap1 - black line, MKI67 knockout Hap1 - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in Hap1 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Intracellular Flow Cytometry analysis of Ramos (Human Burkitt's lymphoma cell line) cells lablling Ki67 with purified ab92742 at 1/150 (red). Cells were fixed with 2% paraformaldehyde. An FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabeled control, cells without incubation with primary and secondary antibodies.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Comparison between RNApII-S2P-/low cells and Ki-67- cells

a : Regulation of Ki-67 and RNApII-S2P during proliferation and quiescence in T98G glioblastoma cells. T98G cells were grown in culture medium containing 10% (v/v) fetal bovine serum (FBS), were induced to become quiescent by serum starvation in medium supplemented with 0.5% (v/v) FBS for 14 days, and then were re-stimulated by being split 1 : 5 into new medium containing 10% (v/v) FBS and cultured for 3 days. The cells were detached from dishes with trypsin-EDTA solution, fixed in 10% (v/v) neutral buffered formalin, and centrifuged. Paraffin sections of the pellet were cut, and expression of Ki-67 and RNApII-S2P was examined by single (brown; a1, a2, a4, a5, a7, a8) or double immunostaining (Ki-67, brown; RNApII-S2P, red; a3, a6, a9). Hematoxylin (blue) was used as a nuclear stain. Ki-67- RNApII-S2P-/low cells (blue cells in the double stained sections) emerged only in the quiescent condition (a6, arrows). Scale bar, 10 μm. b : Single-color immunostaining for Ki-67 (b1) and RNApII-S2P (b2) in serial sections of glioblastoma tissue. Ki-67- tumor cells were frequently found, whereas only a few RNApII-S2P-/low cells (arrows) were observed around necrotic area. N, necrotic area; V, blood vessels. Scale bars, 50 μm.

Ki67 detected using ab92742.

(From Figure S2 of Ishii et al)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Image from Ishii et al PLoS One. 2016 Jan 22;11(1):e0147366. doi: 10.1371/journal.pone.0147366. eCollection 2016. Fig S4.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Chromogenic triple immunostaining for estrogen receptor (ER), progesterone receptor (PgR), and Ki-67 in breast cancer tissue to verify the triple immunostaining detection method.

Panel e :ER⁺ PgR^- Ki-67^- cells were stained red (short arrow), ER^- PgR⁺ Ki-67^- cells were stained blue (black arrowhead), ER⁺ PgR⁺ Ki-67^- cells were stained purple (long arrow), and Ki-67⁺ cells were stained brown (white arrowhead). These colors are easily distinguishable. Scale bars, 25 μm.

Deparaffinized sections were pretreated for antigen retrieval by boiling in antigen retrieval solution, pH 9. Sections were incubated with rabbit monoclonal antibody against Ki67 ab92742 at a 1/1000 dilution. After the reaction with (HRP)-conjugated secondary antibodies color was developed with (DAB) and sections were counterstained with hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

Image from Ishii et al PLoS One. 2016 Jan 22;11(1):e0147366. doi: 10.1371/journal.pone.0147366. eCollection 2016. Fig S4.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Overlay histogram showing Ramos (Human Burkitt's lymphoma cell line) cells stained with unpurified ab92742 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab92742, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Ki67 with ab209897 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on human tonsil.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labeling Ki67 with unpurified ab92742.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunocytochemistry/Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Ki67 with unpurified ab92742 at a dilution of 1/250.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

• ICC/IF

AbReview57409****

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

ab92742 staining Ki67 in human adenocarcinoma cells by ICC/IF (Immunocytochemistry/immunofluorescence).

Cells were fixed with paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS and blocked with 5% serum for 1 hour at 21°C. Samples were incubated with primary antibody (1/1000) for 12 hours at 4°C. A Cy3^® conjugated donkey anti-rabbit IgG polyclonal was used as the secondary antibody at a dilution of 1/200.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

This image is courtesy of an anonymous Abreview.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human squamous cell carcinoma of cervix tissue labeling Ki67 with purified ab92742 at 1/250. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Counterstained with hematoxylin.

Negative control using PBS instead of primary antibody (inset).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic adenocarcinoma tissue labeling Ki67 with unpurified ab92742.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Clone EPR3610 (ab209897) has been successfully conjugated by Abcam. This image was generated using Anti-Ki67 antibody [EPR3610] (Alexa Fluor® 647). Please refer to ab196907 for protocol details.

ab196907 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 1h. The cells were then incubated with ab196907 at 1/100 dilution (shown in red) and ab195887 at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunohistochemical analysis of paraffin-embedded Human bladder cancer labeling Ki67 with ab209897 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleus staining on human bladderr cancer.

Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

ab209897 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), 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 1h. The cells were then incubated with ab209897 at 1μg/ml concentration and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Clone EPR3610 (ab209897) has been successfully conjugated by Abcam. This image was generated using Anti-Ki67 antibody [EPR3610] (Alexa Fluor® 488). Please refer to ab197234 for protocol details.

ab197234 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 1h. The cells were then incubated with ab197234 at 1/100 dilution (shown in green) and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labeling Ki67 with unpurified ab92742.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunocytochemistry/Immunofluorescence analysis of HT-29 (Human colorectal adenocarcinoma cell line) cells labeling Ki67 with purified ab92742 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1 : primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human normal colon tissue labeling Ki67 with unpurified ab92742.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

ab92742 staining Ki67 in wild-type HAP1 cells (top panel) and Ki67 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 1h. The cells were then incubated with ab92742 at 1µg/ml and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labeled in blue with DAPI.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

• WB

Lab

Western blot - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

All lanes:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

false

• WB

Lab

Western blot - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

Blocking and dilution buffer : 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab92742).

All lanes:

Western blot - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (ab209897) at 1/5000 dilution

All lanes:

Ramos (Human Burkitt's lymphoma cell line) cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution

Predicted band size: 358 kDa

Observed band size: 395 kDa

false

• WB

Lab

Western blot - Anti-Ki67 antibody [EPR3610] - BSA and Azide free (AB209897)

This data was developed using the same antibody clone in a different buffer formulation (ab92742).

Western blot : Anti-MKI67 antibody [EPR3610] (ab92742) staining at 1/5000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab92742 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 [EPR3610] (<a href='/en-us/products/primary-antibodies/ki67-antibody-epr3610-ab92742'>ab92742</a>) at 1/5000 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