Early (AnnexinV positive) and Late (AnnexinV-Propidium Iodide (PI) positive) apoptotic cells were assessed with flow cytometry after 12/24/48h from treatment. UW-228, ONS-76, D425med, D283 and D341 cells were seeded at a density of 5.0x104 cells/well, and treated with DSF-Cu++ 150nM and 2uM. Cells were trypsinized and harvested, washed with PBS, incubated with AnnexinV Ab and PI (Invitrogen, Carlsbad, CA, USA), and analyzed. Cell proliferation and cycle were measured by Ki67 and PI expression. Cells were seeded at 2.5x104/well, treated after 24h with 150/300nM DSF-Cu++, and harvested after 24/48h of treatment. Pellets were washed with PBS, fixed and permeabilized with TritonX-100, and stained with anti-Ki67 antibody (Invitrogen, Carlsbad, CA, USA) and PI/RNase solution (FxCycle™, Thermo-Fisher Scientific, Waltham, MA, USA). For cancer stem-cell (CSC) identification, anti-CD133 antibody (BioLegend, San Diego, CA, USA), anti-Nestin (BioLegend, San Diego, CA, USA), and ALDEFLUOR kit (Stem Cell Tech., Durham, NC, USA) were used. Cells were prepared and analyzed with BD FACS Celesta (BD Biosciences; Becton, Dickinson and Company, San Jose, CA, USA), and data analyzed with FlowJo software (FlowJo, LLC).
Anti ki67 antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The Anti-Ki67 antibody is a laboratory reagent used in the detection and quantification of the Ki67 protein, a cellular marker associated with cell proliferation. This antibody can be used in various immunohistochemical and immunocytochemical techniques to identify and analyze cells undergoing active cell division.
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Lab products found in correlation
Anti rabbit hrp dab ihc kit, by Abcam (3 mentions)
Dab substrate kit for peroxidase, by Vector Laboratories (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Cytofix cytoperm fixation and permeabilization solution, by BD (2 mentions)
Warp red chromogen kit, by Biocare Medical (2 mentions)
Vector sg substrate kit, by Biocare Medical (2 mentions)
Neutral buffered formalin, by Merck Group (2 mentions)
Facscelesta, by BD (1 mentions)
Aldefluor kit, by STEMCELL (1 mentions)
Anti nestin, by BioLegend (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Anti her2 antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rat, by Thermo Fisher Scientific (1 mentions)
Anti f4 80 antibody, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
47 protocols using anti ki67 antibody
1
Apoptosis, Proliferation and CSC Analysis
2
Immunohistochemical and Immunofluorescence Assays for HER2, Ki67, and Ovarian Cancer Cell Markers
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The level of HER2 expression was determined by either immunohistochemical analysis or immunofluorescence labeling on frozen or paraffin-embedded tumor tissue sections using an anti-HER2 antibody (cat # 2242, Cell Signaling, Danvers, MA, USA) at 1:100 dilution and standard immunostaining protocols. Secondary antibodies conjugated to horseradish peroxidase (HRP, immunohistochemical) or Alexa fluor-488 (immunofluorescence) were used. HRP was detected with a 3,3'-diaminobenzidine substrate. To determine proliferating cells, an anti-Ki67 antibody (Clone 7B11, Invitrogen, Carlsbad, CA, USA) was used to label the tissue section. Alexa Fluor-555 labeled secondary antibody was used for immunofluorescence imaging. Mouse anti-human CK19 antibody (C-6930, Sigma-Aldrich) was used to identify ovarian cancer cells. Rat anti-mouse CD68 monoclonal antibody (MCA1957, BioRad Laboratories, Inc., Hercules, CA, USA) was used to label macrophages. For immunofluorescence, slides were counter-stained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA).
3
Limbal Stem Cell and Proliferation Assay
4
Immunohistochemical Analysis of Ki-67
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Tumor tissue specimens were fixed in 10% formalin for 2 days and embedded in paraffin to cut the sections. After deparaffinization and dehydration, the sections were incubated in anti-Ki-67 antibody (Invitrogen, Waltham, MA, USA) overnight at 4 °C. The sections were further incubated with an anti-rabbit HRP/DAB IHC kit (Abcam, Cambridge, UK) for 2 h at room temperature after washing.
5
Immunohistochemical Analysis of Ki67 Expression
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The sections were obtained as mentioned above. Sections were incubated in anti-Ki67 antibody (Invitrogen) after de-paraffining and dehydrating. On the next day, sections were washed and probed with an anti-rabbit HRP/DAB IHC kit (Abcam) for 2 h. Images were taken under a microscope.
6
Histological Analysis of Tumor Tissues
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For histology, tumor tissues were extracted from mice. Then, the tissues were embedded in paraffin after being fixed in 10% formaldehyde solution, sectioned (4 μm), and further stained with H&E. For immunohistochemistry, deparaffinization and rehydration of the formalin-fixedparaffin-embedded tissue were performed. Next, the slices were incubated overnight at 4°C with the anti-Ki-67 antibody (Invitrogen, Waltham, MA, USA). Antirabbit HRP/DAB IHC kit (Abcam, Cambridge, UK) was used to stop high background, and all images were captured using a light microscope.
7
Immunofluorescence Staining of COX-2, Tubulin, Ki-67, and F4/80
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Immunofluorescence staining was used to detect COX-2 and tubulin in RAW264.7 cells. The anti-COX-2 antibody (CST, dilution 1:200) was incubated overnight at 4°C and exposed to Alexa Fluor-555-conjugated goat-anti-rabbit (Invitrogen, dilution 1:500) antibody for 1 h at RT. After that, the anti-tubulin antibody (Abcam, dilution 1:200) was also incubated overnight at 4°C and exposed to Alexa Fluor-488-conjugated goat-anti-rabbit (Invitrogen, dilution 1:500) antibody. In addition, immunofluorescence staining was also used to detect Ki-67 and F4/80 in the gum tissue. The anti-Ki-67 antibody (Invitrogen, dilution 1:100) and the anti-F4/80 antibody (Abcam, dilution 1:50) were used as primary antibodies. After the removal of first antibodies, they were exposed to goat anti-mouse IgG H&L (Alexa Fluor® 594, Abcam, United States, dilution 1:500) and Alexa Fluor-488-conjugated goat-anti-rabbit (Invitrogen, dilution 1:500). Nuclei were counterstained with DAPI solution. Images were analyzed using a fluorescence confocal microscope.
8
Protein Expression Analysis in NSCLC
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The total proteins of NSCLC tumors or cells were collected by RIPA lysis buffer (Sangon Biotech). Then, the proteins were separated by 10% Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific). The 5% skimmed milk was added and incubated with primary anti-GAPDH antibody (1:1000, Invitrogen, Carlsbad, CA, USA), anti-β-actin antibody (1:1000, Invitrogen), anti-Ki-67 antibody (1:1000, Invitrogen), anti-matrix metalloprotein-9 (MMP-9) antibody (1:1000, Invitrogen), anti-Cleaved-caspase9 (Cleaved-casp9) antibody (1:1000, Invitrogen) or anti-THBS2 antibody (1:1000, Invitrogen) at 4 °C overnight. Finally, the membranes were incubated with the secondary antibody for 1 h at room temperature. The results were viewed using Kodak film developer (Fujifilm, Japan).
9
Immunohistochemical Staining Evaluation
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Immunohistochemical staining was performed as described previously [14 (link)]. Briefly, IHC was performed using a PV-9001 kit (ZSGB-BIO) following the manufacturer’s instructions. The staining intensity was defined with a four-grade scoring system: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The staining extent was quantified as five value grades: 0 (negative), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The sum of intensity and extent values was defined as the IHC score. The primary antibodies included anti-KDM6A antibody, anti-ARHGDIB antibody, and anti-Ki67 antibody (Invitrogen, PA5–19462).
10
Assessing Cell Proliferation with Ki67
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Ki67 immunofluorescence was performed to detect the effect of 15 h of S4‐2‐2 (6 μM) and DMSO treatment to the cell proliferation index. Briefly, PBS was used to wash the cells at a density of 2 × 105/ml, and then polyfluoroalkoxy was used to fix the cells for 10 min. The cells were then incubated with 0.1% Triton‐X 100 for 10 min. The cells were blocked with 5% bovine serum albumin (BSA) (Sigma) for 1 h and subsequently incubated with anti‐Ki67 antibody (Invitrogen, 1: 200) overnight at 4°C. Following washes, goat anti‐mouse (Invitrogen, 1: 500) was used as a secondary antibody. Nuclei were counterstained with DAPI (Sigma).
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