Four mice were randomly selected from each group for evaluation of Ki-67 protein expression on the metastatic tumors on lungs. Sectioned lung tissue slides were first incubated with a primary Ki-67 antibody (1:3000) (Pharmingen, San Diego, CA) at 4°C overnight, and then with a biotinylated anti-mouse secondary antibody (VECTASTAIN Elite ABS reagent, Vector Laboratories Inc. Burlingame, CA) for 30 min at room temperature. Positive cells were stained brown with 3,3'-diaminobenzidine tetrahydrachloride (DAB) and negative cells were stained blue with hematoxylin [29 (link),30 (link)]. Both positive and negative cells were counted in a given field under the AxioSkop 40 microscope. Results were presented as percentage of positive cells of a given field of tumor cells.
Ki 67 primary antibody
Manufactured by BD
Sourced in United States
The Ki-67 primary antibody is a laboratory reagent used for the detection of the Ki-67 protein, which is a cellular marker for proliferation. This antibody can be used in various immunohistochemical and immunocytochemical applications to identify and quantify proliferating cells.
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Lab products found in correlation
Citrate buffer, by Vector Laboratories (1 mentions)
H 5000, by Vector Laboratories (1 mentions)
Mom blocking buffer, by Vector Laboratories (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Lsrfortessa x 20 cell analyzer, by BD (1 mentions)
Cytofix cytoperm solution, by BD (1 mentions)
Perm wash buffer, by BD (1 mentions)
Discovery xt, by Roche (1 mentions)
Cell conditioning solution, by Roche (1 mentions)
Aperio cs2 digital pathology scanner, by Leica (1 mentions)
Aperio image analysis software, by Leica (1 mentions)
Inhibitor cm, by Roche (1 mentions)
Chromomap dab kit, by Roche (1 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions)
5 protocols using ki 67 primary antibody
1
Quantifying Ki-67 Expression in Metastatic Lung Tumors
2
Ki67 Expression Quantification in Xenograft Tumors
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Immunohistochemistry was done as described previously [19 (link)]. Xenograft tumor tissues were fixed with 4% PFA, dehydrated, and embedded in paraffin. The rehydrated tissue slices were treated by heat-mediated antigen retrieval in citrate buffer (Vector Laboratories, Burlingame, CA). The tissues were made permeable with PBS containing 0.2% TritonX-100, then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase. The tissues were washed with PBS containing 0.05% Tween-20, and incubated for 2 h with blocking solution (MOM blocking buffer, Vector Laboratories, CA). Primary Ki67 antibody (550609, BD Biosciences, San Diego, CA, dilution, 1:2000) was incubated overnight at 4°C, and secondary antibodies were incubated for 1h at room temperature. Signals were amplified with ABC kit (Vector Laboratories) and visualized with 3,3′-diaminobenzidine substrate kit (SK-4105, Vector Laboratories). The tissues was further stained with hematoxylin, then dehydrated, and mounted (H5000, Vector Laboratories). Photograph was taken and Ki67 positive cell percentage was calculated.
3
Quantifying Proliferation in GBM11 Cells
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The GBM11 cells treated with 25 μM Osmi-1 or DMSO for 24 h, and then they were dissociated with trypsin, washed with PBS containing 2% FBS, centrifuged at 1500 RPM for 5 min at 4 °C, and resuspended in 1 mL of FACS buffer (PBS + FBS). In a 96-well plate, 5 × 105 cells were fixed and permeabilized with 100 μL of BD Cytofix/Cytoperm™ solution (BD cat. 554714) for 20 min at 4 °C, and then they were washed 2 times with BD Perm/Wash™ buffer (BD cat. 554723). Next, we proceeded with the incubation with the Ki-67 primary antibody (BD cat. 550609) diluted in BD Perm/Wash™ buffer (1:200) for 20 min at 4 °C in the dark, followed by 2 rounds of washing as described above. Then, the cells were incubated with the FITC anti-IgG mouse secondary antibody diluted in BD Perm/Wash™ buffer (1:200) for 20 min at 4 °C in the dark, and again, they were washed as described above. Finally, the cells were resuspended in 300 μL of FACS buffer and analyzed using a BD LSRFortessa™ X-20 cell analyzer.
4
Quantifying Cellular Proliferation in Formalin-Fixed Tissue
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Four μm-thick sections of formalin-fixed paraffin-embedded (FFPE) explant tissue were stained for the proliferative marker Ki67. Automated immunohistochemistry (IHC) was carried out on a Discovery XT staining module (Roche). Antigen was retrieved by incubating slides in Cell Conditioning Solution (Roche). Endogenous peroxidase activity was reduced by incubating slides in Inhibitor CM (Roche). Slides were incubated in Ki67 primary antibody (Novocastra; 1:100) or Anit-AR primary antibody (clone G122-434, BD) for 1 h at 37°C followed by HRP-conjugated Discovery OmniMaP HRP secondary antibody (Roche) for 30 min. A ChromoMap DAB kit (Roche) was used for detection. Slides were counterstained in hematoxylin and mounted in DPX. Stained slides were imaged using an Aperio CS2 Digital Pathology scanner (Leica). Regions of interest (i.e. epithelial cells) were manually annotated and the percentage of Ki67 positive cells was determined using Aperio Image Analysis software (Leica) in a minimum of four representative 40× magnification regions containing a total of at least 1000 nuclei. Positive control tissue for Ki67 is shown in Supplementary Figure S8 alongside no primary control staining. Serial sections were also stained for p63 (basal cell marker identifying benign regions) and AMACR (cancer cell marker) to define regions of cancer (Supplementary Figure S9 ).
5
Proliferation Dynamics of ISC3D-hIO
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The ISC3D-hIO was plated onto 4-well chamber slides (Nunc, Cat No. 177437, Thermo Fisher Scientific Inc., Waltham, MA, USA). After cell attachment in full medium for 2 days, the ISC3D-hIO was grown for 4 days in each growth factor-depleted medium. The ISC3D-hIO was then grown for 24 h in each medium containing 10 μM EdU (Invitrogen, Cat No. C10640). EdU-incorporated ISC3D-hIO was fixed in 4% paraformaldehyde in Dulbecco’s phosphate buffered saline (without Ca2+ and Mg2+). The EdU-positive cells were labelled with the fluorescent dye picolyl azide probe followed by the manufacturer’s instructions. For co-staining, samples were labelled by KI67 primary antibody (1:100, BD Bioscience, Cat No. 556003, BD Bioscience, Becton, NJ, USA), which was diluted in 4% bovine serum albumin (BSA, Bovogen Biologicals, Cat No. BSA100, Victoria, Australia) in PBS and DAPI (4’,6-diamidino-2-phenylindole dihydrochloride, Invitrogen, Cat No. D1306) for labelling nuclei. The KI67+ and BrdU+ cells were counted independently in three ISC colonies.
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