Immunofluorescence assays were performed as described previously36 (link). The primary antibody, anti-Ki67 (sc-23900), was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-FHL2 (ab12327) was obtained from Abcam (Cambridge, UK). Anti-β-catenin (#8480) was obtained from Cell Signaling Technology (Danvers, MA, USA). The fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse (D110081-0100) and Cy3-conjugated donkey anti-rabbit (D110052-010) secondary antibodies were obtained from Sangon Biotech (Shanghai, China).
Anti ki67 sc 23900
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Ki67 (sc-23900) is a primary antibody product offered by Santa Cruz Biotechnology. It is designed to detect the Ki-67 protein, which is a well-established marker of cellular proliferation.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Anti β catenin 8480, by Cell Signaling Technology (1 mentions)
Ab211777, by Abcam (1 mentions)
Buthionine sulfoximine, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Anti β actin, by Transgene (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti oct4, by Cell Signaling Technology (1 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Polybrene, by Yeasen (1 mentions)
Bafilomycin a1, by Sangon (1 mentions)
Pepstatin a, by Sangon (1 mentions)
Anti beclin 3495, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti mouse, by Boster Bio (1 mentions)
Pyronin y, by Merck Group (1 mentions)
Anti atg5, by Cell Signaling Technology (1 mentions)
Anti sqstm1 p62, by Cell Signaling Technology (1 mentions)
Anti β tubulin, by Transgene (1 mentions)
Anti vimentin, by Boster Bio (1 mentions)
9 protocols using anti ki67 sc 23900
1
Immunofluorescence Assay Protocol for Cell Markers
2
Immunofluorescence Staining of Cultured Cells
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The cells cultured in 6-well dishes were fixed in 4% paraformaldehyde and permeabilized with 0.25% Triton X-100, followed by blocking with 3% BSA in PBS. Then cells were probed with the primary antibody in 3% BSA for 1 h at 4 °C and the secondary antibody in 3% BSA for 30 min at room temperature. A drop of Vectashield mounting medium with 49, 6-diamidino-2-phenylindole (DAPI; C1006, Beyotime, Shanghai, China) was placed on the microscope slide, and the coverslip was sealed with nail polish in a way that the cells were in contact with the mounting medium. The staining signal was then observed through the microscope. The primary antibodies used were: anti-NR2F2 (ab211777, Abcam, Cambridge, UK), and anti-Ki67 (sc-23900, Santa Cruz, Dallas, TX, USA).
3
Comprehensive Cell Autophagy Assay
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H2DCFDA, rapamycin, pyronin Y, and buthionine sulfoximine were obtained from Sigma-Aldrich Co. (USA). Bafilomycin A1 and pepstatin A were obtained from Sangon Biotech Co. (China). D-Luciferin was obtained from Goldbio Co. (USA). Polybrene, Hoechst 33342, and propidium iodide (PI) were obtained from Yeasen Biotech Co. (China). Transfection reagents DharmaFECT, Lipofectamine 2000, and Alexa Fluor 488 and 567 secondary antibodies were obtained from Thermo Fisher Scientific Inc. (USA, 1:500). Anti-LC3 (#3868, 1:1000), anti-Beclin (#3495, 1:1000), anti-ATG5 (#12994, 1:1000), anti-Oct-4 (#2750, 1:1000), and anti-p62/SQSTM1 (#7695, 1:1000) antibodies were obtained from Cell Signaling Technology (USA). Anti-RB1CC1 (sc-22709, 1:100), anti-Ki-67 (sc-23900, 1:100), and HRP-conjugated goat anti-rabbit secondary antibodies were obtained from Santa Cruz Biotechnology (USA). Anti-vimentin, HRP-conjugated goat anti-mouse, and rabbit anti-goat secondary antibodies were obtained from Boster Co. (China). Anti-β-tubulin (1:1000) and anti-β-actin (1:1000) antibodies were obtained from Transgene Biotech Co. (China). Anti-NRF2 (WL02135, 1:2000), anti-NOTCH1 (WL01991, 1:500), and anti-Lamin B (WL01775, 1:500) antibodies were obtained from Wanleibio Co. (China).
4
Immunohistochemical Analysis of Ki-67 and ILK
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A certain amount of citrate buffer (pH=6.0) was added to the sections. After being heated by medical microwave for antigen retrieval, the sections were allowed to cool down to indoor temperature. Each section was added with 1 drop of 3% H2O2 and incubated for 10min at indoor temperature. Then, they were incubated with goat serum for 15min at indoor temperature and the serum was discarded. Consecutive 4-μm-thick sections were analyzed using primary antibodies against ki-67 and anti-ILK (1:100,1:500) (anti-ki67, sc-23900; anti-ILK, sc-20019, Santa Cruz Biotechnology, CA, USA) and incubated overnight at 4˚C. Flushed with PBS, sections were added with the secondary antibodies (Abcam, Cambridge, MA, USA) which were labeled with horseradish peroxidase and incubated for 30-40min at 4°C. Cells were dyed and flushed with PBS. Cells that were sealed by epoxy resin and anti-quench reagent were subjected to observation under light microscope.
5
Xenograft Protein Expression Analysis
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Immunohistochemistry analysis was conducted on formalin-fixed paraffin-embedded tissue sections, as previously reported (Mitsuda et al., 2018 (link)). The following antibodies were used in this study: anti-survivin (sc-17779; Santa Cruz Biotechnology), anti-RUNX1 (A-2; Santa Cruz Biotechnology), and anti-Ki67 (sc-23900; Santa Cruz Biotechnology) antibodies for xenograft experiments. Tissue section images were captured using a BZ-X700 all-in-one fluorescence microscope (Keyence, Japan).
6
Immunohistochemical Analysis of Cell Markers
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Immunohistochemistry analysis was performed as described previously40 (link), using a super-sensitive horseradish peroxidase immunohistochemistry kit (Sangon Biotech, Shanghai, China); the anti-Ki67 (sc-23900, Santa Cruz), anti-IGF1R (D163034, BBI), and anti-IRS1 (D120888, BBI) antibodies were used.
7
Tissue Preservation and Immunofluorescence Analysis
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Excised organs were fixed with 4% paraformaldehyde (PFA) overnight, washed with PBS three times for 5 min each, and dehydrated with 10% sucrose overnight. Dehydrated organs were embedded in an optimal cutting temperature (OCT) compound (TISSUE-TEK 4583, Sakura Finetek Inc., Torrance, CA, USA) and sliced into sections (10 µm thick) using a cryotome (HM 525; Thermo Fisher Scientific, Waltham, MA, USA). After hydration, the sections were fixed and stained with hematoxylin (HHS16, Sigma Aldrich, St Louis, MO, USA) and eosin (HT110116, Sigma Aldrich) according to a standard protocol.
The fixed sections were permeabilized with 0.1% Triton X-100. After incubation with 1% BSA for 1 h, anti-Ki67 (sc23900, Santa Cruz Biotechnology, Dallas, TX, USA) and anticaspase-3 (ab44976, Abcam) antibodies were added, and the samples were incubated overnight. After washing, secondary antibodies conjugated with Alexa Fluor 488 (1 : 1000 dilution) and Alexa Fluor 555 (1 : 1000 dilution) were applied for 0.5 h, followed by DAPI staining for 10 min. The sections were mounted in mounting solution (Dako Cytomation) and visualized by confocal fluorescence microscopy (LSM 880 META, Zeiss, Germany).
The fixed sections were permeabilized with 0.1% Triton X-100. After incubation with 1% BSA for 1 h, anti-Ki67 (sc23900, Santa Cruz Biotechnology, Dallas, TX, USA) and anticaspase-3 (ab44976, Abcam) antibodies were added, and the samples were incubated overnight. After washing, secondary antibodies conjugated with Alexa Fluor 488 (1 : 1000 dilution) and Alexa Fluor 555 (1 : 1000 dilution) were applied for 0.5 h, followed by DAPI staining for 10 min. The sections were mounted in mounting solution (Dako Cytomation) and visualized by confocal fluorescence microscopy (LSM 880 META, Zeiss, Germany).
8
Characterization of CSPG4-expressing Cells
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The mouse anti-CSPG4 monoclonal antibody clone 9.2.27 (#554275) was purchased from BD Biosciences. The donkey anti-mouse Alexa Fluor 488® (#A-21202) and goat anti-mouse Alexa Fluor 568® (#A-11004) secondary IgG antibodies were obtained from Life Technologies Corporation. Anti-CD271 (LNGFR) APC-conjugated human monoclonal antibody (#130-091-884) was purchased from MACS Miltenyi Biotec. The APC mouse IgG1, κ isotype control (FC) antibody (#400122) was obtained from BioLegend, Inc. The mouse monoclonal antibodies anti-Ki67 (sc-23900), as well as mAbs against CSPG4: Anti-NG2 clone G-9 (sc-166251) and anti-NG2 clone LHM 2 (sc-53389), were purchased from Santa Cruz Biotechnology, Inc. The primary anti-human CSPG4 antibody clone 132.38 (#ab50009) was purchased from Abcam. Control mouse IgG (#I8765) was obtained from Sigma-Aldrich (Merck KGaA). The mouse monoclonal antibodies anti-p44/42 MAPK (Erk1/2) clone L34F12 (#4696), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) clone E10 (#9106), anti-Akt (pan) clone 40D4 (#2920), anti-phospho-Akt (Ser473) clone 587F11 (#4051) and anti-β-actin clone 8H10D10 (#3700) were purchased from Cell Signaling Technology, Inc. Anti-mouse IgG, HRP-linked secondary antibody (#7076) was obtained from Cell Signaling Technology, Inc.
9
Regulation of Apoptosis and Proliferation in Inflammatory Cells
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RPMI 1640, penicillin and streptomycin solution, and fetal bovine serum (FBS) were purchased from Life Technologies Inc. TAK-242, an inhibitor of TLR4 (TLR4i), was obtained from Calbiochem (San Diego, CA, USA), respectively. Imatinib mesylate, a selective tyrosine kinase inhibitor, was obtained from Sigma. Anti-Bcl-2 antibodies (554160) were obtained from BD Biosciences. Anti-TLR4 (sc-10741), anti-ERK1/2 (sc-154), anti-β-actin (sc-47778), anti-pro-caspase 9 (sc-17784), anti-pro-caspase 3 (sc-7272), anti-phospho-AKT (sc-109903), anti-AKT (sc-8312), anti-PDGFRα (sc-398206), anti-S100A8 (sc-20174), anti-S100A9 (sc-20173), and anti-Ki-67 (sc-23900) antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz). Z-DEVD-fmk (60332), anti-phospho-STAT3 (9131), anti-STAT3 (9139), anti-cleaved caspase 9 (9501), anti-cleaved caspase 3 (9664), anti-AIF (4642), anti-Bax (2772), anti-Mcl-1 (4572), anti-phospho-ERK1/2 (9101), anti-phospho-p38 MAPK (9211), anti-p38 MAPK (9212), anti-phospho-JNK (9251), anti-JNK (9252), anti-rabbit IgG-HRP (7074), and anti-mouse IgG-HRP (7076) antibodies were procured from Cell Signaling Technology. Anti-phospho-Bad (9296) and anti-Bad (9292) antibodies were purchased from New England Biolabs. Anti-phospho-PDGFRα antibodies (ab5460) for immunohistochemistry were obtained from Abcam.
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