The organoids were fixed with 10% neutral buffered formalin, embedded in paraffin, and sectioned onto slides. Slides were subjected to hematoxylin and eosin (H&E) staining or immunohistochemistry (IHC) staining following standard protocols. For IHC staining, the samples were incubated with the primary anti-Ki67 antibody (Cell Signaling Technology). Images were then taken with an Olympus microscope (100×).
Anti ki67 primary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-Ki67 primary antibody is a laboratory reagent used for the detection and quantification of the Ki67 protein, a well-established marker of cellular proliferation. This antibody can be used in various immunodetection techniques, such as immunohistochemistry and immunocytochemistry, to assess the proliferative status of cells.
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6 protocols using anti ki67 primary antibody
1
Assessing Organoid Proliferation by IHC
2
Immunohistochemical Analysis of Xenograft Tumors
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The xenograft tumors were formalin-fixed and paraffin-embedded, sliced into 4µm-thick sections. The xenograft tumors were formalin-fixed and paraffin-embedded, sliced into 4 µm-thick sections. Graded ethanol was used to rehydrate the sections after deparaffinization in xylene at room temperature. After washing with PBS, the sections were placed in 3% hydrogen peroxide for 20 min to inhibit endogenous peroxidase, followed by antigen retrieval by heating for 30 min in a microwave. After being blocked with 10% goat sera, the tissue sections were incubated with primary anti-Ki67 antibody (1:500, Cell Signaling Technology, MA, USA) and at 4°C overnight. After being washed with PBS and incubated with secondary antibody for 30 min at 37°C, 3,′3-Diaminobenzidine tetrahydrochloride (DAB) was applied as a chromogen, and the sections were counterstained with hematoxylin.
3
Quantifying Cellular Proliferation via Ki67
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Tissue sections were prepared and treated as described for immunofluorescence staining. Staining was performed with anti-Ki67 primary antibody (9027, Cell Signaling) at 4 °C overnight followed by biotin-conjugated polyclonal swine anti-rabbit secondary antibody (E0353, Agilent Technologies, Santa Clara, CA, USA) and phosphatase-conjugated streptavidin (Seracare Life Sciences, Milford, CT, USA). Signals were visualized by HistoMark® RED (Seracare Life Sciences) and hematoxylin counterstaining (Sigma-Aldrich). For quantification, Ki67-positive cells were counted per high power field (HPF = 0.159 mm2, 10 HPF counted). The mean of Ki67-positive cells was calculated and expressed in percent.
4
Immunohistochemical Analysis of Arterial Remodeling
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Immunohistochemical staining was conducted as previously described [5 (link)]. After fixed with 4% paraformaldehyde, the arteries were perfused with saline and embedded in paraffin. The artery sections (4–5 μm) were obtained at 80-μm intervals and then stained with hematoxylin and eosin (H&E; Solarbio). Finally, the areas of intima and media were measured by using Image-Pro Plus software. For immunohistochemical staining, artery sections were incubated with anti-Ki-67 primary antibody (cat 9449 T; Cell Signaling Technology) after being blocked (4°C, overnight). The sections were then incubated with a secondary antibody (Proteintech, Rosemont, USA) and counterstained with Mayer hematoxylin. Five artery sections per sample were collected for staining.
5
Immunohistochemical Analysis of Ki67
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The paraffin-embedded sections were stained with hematoxylin and eosin (H&E) using a Leica Autostainer XL (ST5015, Leica Microsytem) for tissue histological analysis. Expression of Ki67 was investigated by immunohistochemical analysis using anti-Ki67 primary antibody (Cell Signalling) and an HRP-conjugated anti-rabbit secondary antibody (Jackson Immunresearch) following DAB enhancement (Sigma). The prepared specimens were imaged with a brightfield microscope. Sections without primary antibodies were used as negative controls and showed no immunoreactivity.
6
Immunohistochemical Analysis of Tumor Markers
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Formalin-fixed, paraffin-embedded tissues of transplanted tumors were sectioned at 4-mm thickness and analyzed for anti-Foxq1 primary antibody (Abcam Ltd, Cambridge, UK), anti- Ki-67 primary antibody, and anti- PCNA primary antibody (Cell Signaling Technology, Inc.USA). Visualization was achieved using the EnVisionþ peroxidase system (Dako). A sample was considered positive if more than 50% of the tumor cells retained nuclear staining, and 5 fields were randomly selected according to semiquantitative scales. The intensity of staining was scored manually: negative (-), weak positive (+), medium positive (++), strong positive (+++). Data recording and analysis by 2 independent experienced pathologists, and only tumor cells were scored.
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