Cryopreserved sections were blocked with 5% BSA/5% horse serum (Sigma) and incubated overnight with primary antibodies: rabbit anti-human K5 (1:500, Biolegend), mouse anti-human K10 (1:500, DAKO), mouse anti-human Col IV (1:500, Sigma), rabbit anti-human CD29 (1:200, GeneTex, Irvine, USA), mouse anti-human Ki67 (1:100, DAKO), mouse anti-human Vimentin (1:200, DAKO), rabbit anti-human pan cytokeratin (1:100, Novus Biological, Littleton, USA) or mouse anti-human α5 chain Laminin (1:500, Millipore, Burlington, USA). Slides were washed and incubated with Alexa Fluor-conjugated mouse or rabbit secondary antibodies (BD Biosciences, Columbus, USA). Sections were imaged on a Nikon Ti-E microscope. Ki67 staining was quantitated for fluorescent nuclei with the ‘Tissue Analysis Cell Counter’ macro using FIJI software (NIH). Ki67 and DAPI nuclear signal thresholds were adjusted to minimise nuclear segmentation or fusion and verified by manual count.
Mouse anti human ki67
Manufactured by Agilent Technologies
Sourced in United States
The Mouse anti-human Ki67 is a laboratory reagent used to detect the Ki67 protein, a cellular marker for proliferation. It is a primary antibody that binds specifically to the Ki67 antigen expressed in the nuclei of proliferating cells.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti human vimentin, by Agilent Technologies (1 mentions)
Ti e microscope, by Nikon (1 mentions)
Harris hematoxylin, by Merck Group (1 mentions)
Clone 1a4, by Agilent Technologies (1 mentions)
Fitc phalloidin, by Merck Group (1 mentions)
Rabbit anti human vwf, by Abcam (1 mentions)
Rabbit anti human β catenin, by Merck Group (1 mentions)
Goat antihuman ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Mouse anti human cd31, by Agilent Technologies (1 mentions)
Dako real detection system, by Agilent Technologies (1 mentions)
Lsm 510 confocal laser scanning microscope, by Zeiss (1 mentions)
Novocastra antigen retrieval solution ph6 or ph 9, by Leica (1 mentions)
Dako blocking solution, by Agilent Technologies (1 mentions)
Donkey anti mouse cy5, by Jackson ImmunoResearch (1 mentions)
Prolong gold dapi, by Thermo Fisher Scientific (1 mentions)
510 meta confocal microscope, by Zeiss (1 mentions)
Donkey anti mouse conjugated with tritc, by Jackson ImmunoResearch (1 mentions)
Donkey anti rabbit conjugated with cy5, by Jackson ImmunoResearch (1 mentions)
7 protocols using mouse anti human ki67
1
Immunostaining of Cryopreserved Tissue Sections
2
Immunohistochemical Analysis of Ki-67 in Tumor Sections
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Tumor sections (5 μm) were blocked with 5% BSA in PBS and incubated with primary antibodies against Ki-67 (mouse anti-human Ki-67; 1 : 200, clone 1A4, Dako, Gostrup, Denmark). This step was followed by the addition of alkaline phosphatase conjugated polymer Mach 2 (Biocare Medical, Concord, USA) and visualized by Fast Blue BB/Naphthol-AS-MX-Phosphate, resulting in a blue/purple-colored precipitate. Hematoxylin and eosin staining was routinely preformed. Briefly, sections were deparaffinized in xylene I, II and III, washed and stained in hematoxylin (Harris Hematoxylin, Sigma) followed by eosin staining. In the next step they were dehydrated by washing in ethanol and mounted with DPX mountant and coverslip. Sections were imaged using Olympus microscope IX81 connected to Leica 200 scientific camera.
3
Immunofluorescence Staining Protocol
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Whole constructs were fixated in paraformaldehyde (4%), for 20 min, and then permeabilized with 0.3% Triton X-100 (Bio Lab Ltd.), for 10 min. Constructs were then washed with PBS and immersed overnight in BSA solution (5%; Millipore). Samples were then incubated with goat antihuman VE-cadherin (1:100; Santa Cruz), mouse antihuman Yes-associated protein (YAP) (1:100; Santa Cruz), mouse antihuman NG2 (1:100; Santa Cruz), rabbit antihuman vWF (1:150; Abcam), rabbit antihuman β-catenin (1:100; Sigma-Aldrich), or mouse antihuman Ki67 (1:20, DAKO) antibodies, overnight, at 4°C. Constructs were then treated with Cy3-labeled (1:100; Jackson Immunoresearch Laboratory), Cy5-labeled (1:100; Jackson Immunoresearch Laboratory), or Alexa-488 (1:400; ThermoFisher Scientific) antibodies, mixed with DAPI (1:1000; Sigma-Aldrich), for 2 h, at room temperature. For the phalloidin staining, constructs were treated with FITC phalloidin (1:100; Sigma-Aldrich) and DAPI for 20 min. For the mitomycin experiment, mitomycin-treated cells and control cells without mitomycin were fixated in paraformaldehyde (4%), for 20 min on day 10 of culture, and then incubated in a 30% (wt/vol) sucrose solution overnight, embedded in optimal cutting temperature compound (Tissue-Tek) and frozen for subsequent cryosectioning (5–20 µm). Standard protocols were used for H&E and Masson’s trichrome staining of the sections.
4
Immunohistochemical Analysis of Liver Organoids
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Immunoperoxidase-based immunohistochemistry was performed to stain 2 μm sections of paraffin-embedded liver organoids. The following primary antibodies were used: mouse anti human CK8/18 (monoclonal, concentrated, pH6; BD Pharmingen, San Jose, CA, USA), rabbit anti human Vimentin (monoclonal, 1:400, pH6; Thermo scientific, Waltham, MA, USA), mouse anti human CD31 (monoclonal, 1:500, pH9; DAKO), mouse anti human E-Cadherin (monoclonal, 1:50, pH9, Thermo scientific), mouse anti human Ki67 (monoclonal, 1:800, pH6, DAKO); Immunohistochemistry was performed as described in detail elsewhere [13 (link)] using the following chemicals and reagents: antigen retrieval in Novocastra antigen retrieval solution pH6 or pH 9.0 (Leica, Wetzlar, Germany); blocking of endogenous peroxidase (DAKO blocking solution, DAKO, Hamburg, Germany); detection of bound antibodies by the immunoperoxidase/DAB-based DAKO REAL detection system (DAKO). For immunofluorescence staining, liver organoid sections were incubated overnight (37°C) with primary antibodies, mouse anti-E-Cadherin (1:100) and rabbit anti-albumin (1:500) or non-immune IgGs diluted in blocking buffer, washed and incubated with secondary antibodies in blocking buffer for 1 h at room temperature. Nuclear stain was Draq5 (diluted 1:1000 in PBS). Sections were analysed in a Zeiss LSM 510 confocal laser scanning microscope (Carl Zeiss, Germany).
5
Immunohistochemical Staining of Ki67 and CD117
6
Immunofluorescence Assay for Maspin, Ki-67, and Lamin C
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For immunofluorescence experiments, cells were plated on glass coverslips, fixed in 4% paraformaldehyde for 20 minutes and permeabilized with 0.5% Triton X-100 in PBS. Fixed cells were incubated in primary antibody solution overnight at 4°C, washed with PBS, and incubated for 1 hour with secondary antibody solution at room temperature and washed again in PBS. Coverslips were mounted on glass slides using DABCO mounting medium (Fluka) with DAPI. Staining was visualized on a Zeiss 510 Meta confocal microscope using the 63X objective. Primary antibodies used for staining were mouse anti-human maspin (BD Pharmingen, 1:25), mouse anti-human Ki-67 (DakoCytomation, 1:50), rabbit anti-human lamin C (a kind gift from prof. C.J Hutchison, 1:20). Secondary antibodies were donkey anti-mouse conjugated with TRITC and donkey anti-rabbit conjugated with Cy5 (both from Jackson ImmunoResearch). After 24 hours to 120 hours following transfection, EGFP-expressing cells were counted for expression of Ki-67 antigen in five to ten fields of view and the Ki-67-positive subpopulation was calculated as the percentage of all transfected cells.
7
Immunohistochemical Analysis of Tumor Samples
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Consecutive cryosections (4 μm) of each tumor were fixed in acetone (10 min, RT) and incubated in H2O2 (10 min, RT, 0.03%) to block endogenous peroxidase activity. Subsequently, slides were incubated with either mouse anti-rat CD31 (1:100,2 h, Becton Dickinson®, Heidelberg, Germany), rabbit anti-human Caspase-3 (1:50; 2 h, Cell Signaling, Boston, MA, USA), mouse anti-human EpCAM VU1D9 (1:2000; 1 h, Cell Signaling, Boston, MA, USA) or mouse anti-human Ki67 (1:800;1 h, Dako®, Hamburg, Germany). Thereafter, sequential incubations with biotinylated anti-mouse, rat-adsorbed (for CD31, EpCAM and Ki67), anti-rabbit secondary antibody (for Caspase 3), and peroxidase-labelled avidin-biotin-peroxidase complex were conducted (Vector Lab. Inc.®, Burlingame, CA, USA). Amino-ethyl-carbazole (AEC) peroxidase substrate was used for the detection of antigen/antibody complexes indicated by red-brown staining. Counter-staining was achieved with hematoxylin (blue). Negative controls were conducted simultaneously using respective mouse/ rabbit isotype-control antibody (Cell Signaling). Finally, sections were mounted in Kaiser’s glycerol gelatin for subsequent analysis.
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