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)

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.

Alexa Fluor^® 488 (ab197234) and Alexa Fluor^® 647 (ab196907) conjugated versions are available for this clone.

Anti-Ki67 antibody [EPR3610] (ab92742) at 1/5000 dilution (purified) + Ramos (Human Burkitt's lymphoma cell line) cell lysate at 20 µg

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

Predicted band size: 359 kDa
Observed band size: 395 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

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 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

Alexa Fluor^® 488 (ab197234) and Alexa Fluor^® 647 (ab196907) conjugated versions are available for this clone.

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).

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.

Immunocytochemistry 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).

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human normal colon tissue labeling Ki67 with unpurified ab92742. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colonic adenocarcinoma tissue labeling Ki67 with unpurified ab92742. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labeling Ki67 with unpurified ab92742. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Anti-Ki67 antibody [EPR3610] (ab92742) at 1/500 dilution (unpurified) + HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

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

Predicted band size: 359 kDa
Observed band size: 395 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cervical carcinoma tissue labeling Ki67 with unpurified ab92742. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

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

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.

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