Anti-Ki67 Antibody Picoband® (monoclonal, 5C7)

Western blot analysis of Ki67 using anti-Ki67 antibody (M00254-9).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human K562 whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-Ki67 antigen affinity purified monoclonal antibody (Catalog # M00254-9) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for Ki67 at approximately 358 kDa. The expected band size for Ki67 is at 358 kDa.

IHC analysis of Ki67 using anti-Ki67 antibody (M00254-9).
Ki67 was detected in a paraffin-embedded section of human lung cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-Ki67 Antibody (M00254-9) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

Effects of upregulation and downregulation of MALAT1 on U87 and U251 cell proliferation. ( a and b ) Cellular proliferation of untransfected or transfected U87 and U251 cells was measured using a CCK-8 assay daily for 3 days. ( c ) Cellular proliferation of untransfected or transfected U87 and U251 cells was measured by testing the expression of Ki-67. ( d and e ) The percentage of Ki-67-positive cells was calculated. Results are expressed as mean±S.D. from three independent experiments ( P <0.01). ( f ) Untransfected or transfected U87 and U251 cells were stained by propidium iodide and analyzed using flow cytometry. ( g and h ) The percentage of cells in the G0/G1, S and G2/M phases of the cell cycle was calculated. Results are expressed as mean±S.D. from three independent experiments ( P <0.01). Abbreviations: CCK-8, Cell Counting Kit-8; MALAT1, metastasis-associated lung adenocarcinoma transcript 1; S.D., standard deviation
Index in PubMed under a CC BY license. PMID: 26938295

Effects of overexpression of MALAT1 on proliferation in vivo . ( a and b ) Overexpression of MALAT1 reduced the growth of glioma in a subcutaneous glioma nude model. ( c and d ) MALAT1 expression in subcutaneous and incratranial tumors ( P <0.01). ( e ) Immunohistochemistry showed that overexpression of MALAT1 reduced the expression of Ki-67 and MMP2 in intracranial xenograft of U87. ( f and g ) Western blotting showed that overexpression of MALAT1 reduced the expression of P-ERK ( P <0.01). ( h and i ) The percentage of Ki-67- and MMP2-positive cells, respectively, calculated from densitometry immunohistochemistry signaling. Results are expressed as mean±S.D. from three independent experiments ( P <0.01). ( j ) Kaplan–Meier survival curves for nude mice implanted intracranially with U87 and its MALAT1 transfectants. MALAT1, metastasis-associated lung adenocarcinoma transcript 1
Index in PubMed under a CC BY license. PMID: 26938295

Cripto-1 gene silencing suppresses cell proliferation in vivo . (a) representative mouse bearing tumors (up was in CNE-2 control group, down was in CNE-2/GFP + /Cripto-1 - group). ( b) external whole-body fluorescence images of the same mouse. ( c) the external images of xenotransplant tumors (top was in CNE-2/GFP + /Cripto-1 - group, bottom was in CNE-2 control group). ( d) H&E stains of xenotransplant tumors, (×400). (e) expression of Ki67 in xenotransplant tumors of CNE-2 control group (×400). (f) expression of Ki67 in xenotransplant tumors of CNE-2/GFP + /Cripto-1 - group (×400).
Index in PubMed under a CC BY license. PMID: 19732464

IHC analysis of Ki67 using anti-Ki67 antibody (M00254-9).
Ki67 was detected in a paraffin-embedded section of human lymphomas tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-Ki67 Antibody (M00254-9) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

IHC analysis of Ki67 using anti-Ki67 antibody (M00254-9).
Ki67 was detected in a paraffin-embedded section of human liver cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-Ki67 Antibody (M00254-9) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

IHC analysis of Ki67 using anti-Ki67 antibody (M00254-9).
Ki67 was detected in a paraffin-embedded section of human esophageal squamous carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-Ki67 Antibody (M00254-9) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

IHC analysis of Ki67 using anti-Ki67 antibody (M00254-9).
Ki67 was detected in a paraffin-embedded section of human cervical cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-Ki67 Antibody (M00254-9) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

IF analysis of Ki67 using anti-Ki67 antibody (M00254-9).
Ki67 was detected in an immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL mouse anti-Ki67 Antibody (M00254-9) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Flow Cytometry analysis of Jurkat cells using anti-Ki67 antibody (M00254-9).
Overlay histogram showing Jurkat cells stained with M00254-9 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-Ki67 Antibody (M00254-9, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

IHC analysis of Ki67 using anti-Ki67 antibody (M00254-9).
Ki67 was detected in a paraffin-embedded section of HepG2 subcutaneous xenograft in nude mouse tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:200 mouse anti-Ki67 Antibody (M00254-9) overnight at 4°C. Two step IHC kit was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.