Tubulin-specific mAb DM1α (Sigma-Aldrich), anti-centromere antibody (CREST), anti-CC-3 antibody (Cell Signaling), anti-Ki67-antibody (Ventana), anti-γ-H2AX-antibody (Novus Biologicals), GFP-specific antibody (William Wickner). Antibodies were used at dilutions of 1:1,000 or 1:10,000 (for GFP-specific antibody).
Anti ki67 antibody
Manufactured by Roche
Sourced in United States, Germany
The Anti-Ki67 antibody is a laboratory reagent used in immunohistochemistry and flow cytometry applications. It binds to the Ki67 protein, which is a cellular marker associated with proliferation. This antibody can be used to detect and quantify the presence of proliferating cells within a sample.
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Lab products found in correlation
Deadend colorimetric tunel system, by Promega (2 mentions)
Axioplan 2 microscope, by Zeiss (2 mentions)
Anti cc3 antibody, by Cell Signaling Technology (1 mentions)
Anti γ h2ax antibody, by Novus Biologicals (1 mentions)
Dm2700 m bright field microscope, by Leica (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Discovery auto stainer, by Roche (1 mentions)
Anti cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
10 protocols using anti ki67 antibody
1
Immunofluorescence Microscopy Protocol
2
Histological Analysis of Apoptosis
3
Apoptosis and Proliferation in Mouse Brains
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Brains were collected from the mice 3 h after completion of the last treatment (n = 2 for each treatment). Paraformaldehyde-fixed brains were paraffin-embedded and sectioned (10 µm) for hematoxylin and eosin (HE) and anti-Ki67 antibody (2 µg/mL) (Ventana, Tucson, AZ, USA) staining. To assay the apoptotic response to treatment, TUNEL staining was performed using the DeadEnd Colorimetric TUNEL system (Promega, Madison, WI, USA) according to the manufacturer’s protocol for paraffin-embedded tissues.
4
Quantifying Skin Cell Proliferation
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Formalin-fixed skin sections from saline- and rIL-31-treated mice were prepared and stained with anti-Ki67 antibody (Clone 30–9, Ventana, Tucson, AZ, USA) as detailed in our previous study [18 (link)]. The co-immunostained images were captured using a Leica DM2700 M bright-field microscope (Leica Microsystems).
5
Quantifying Proliferation in Lung Tissue
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Frozen lung tissue sections were immunostained with anti-Ki67 antibody (Roche) and examined using a Zeiss Axioplan 2 microscope (Zeiss). The percentage of proliferating PMT-derived cells was determined by enumerating GFP+ Ki67+ cells among total GFP+ cells in ≥7 random 20x microscopic fields for each sample. To confirm the specificity of Ki67 immunostaining, paraffin embedded sections or WT alveolar macrophages isolated by BAL and adherence were also stained with Ki67 and examined by light microscopy.
6
Quantifying Proliferation in Lung Tissue
7
Immunohistochemical Analysis of Ki67 in Tissues
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Spleen and lymph nodes were fixed overnight at room temperature in PBS containing 4% formaldehyde and stored in 70% ethanol. Immunohistochemistry with antibodies directed against Ki67 was performed by the GIGA immunohistology platform using established protocols [60 (link)]. Briefly, tissue sections were subjected to heat-induced epitope retrieval using a pressure cooker, rinsed in water and incubated in 3% hydrogen peroxide in methanol for 30 min. After washing in PBS, non‐specific binding was reduced by incubation with normal goat serum. Then, samples were labeled with anti-Ki67 antibody (# 790–4286, Roche) for 1 hour at room temperature, washed twice with PBS and incubated with an anti-rabbit peroxidase conjugate (# K4003, Dako) for 30 minutes. Samples were revealed with diaminobenzidine tetrahydrochloride (DAB), washed with distilled water and observed under light microscopy with a 40× objective. Quantification of scanned images was performed with QuPath (0.1.2).
8
Histological Analysis of Ki67 Expression
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From each Formalin-fixed parafinembedded block, we cut two consecutive sections (3.5 μm): One for H&E staining and one for staining with the Anti-Ki67 antibody. For H&E staining, we used undiluted Mayer’s hematoxylin and 0.5% eosin. For IHC, we used Anti-Ki67 antibody (Roche, United States), 3,3’-diaminobenzidine as chromogen, and Mayer’s hematoxylin as a counterstain with a 1:10 dilution.
9
Immunohistochemistry Protocol for RUNX3 and Ki-67 Detection
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Immunohistochemistry was performed with the indirect enzyme-labeled antibody method, as described previously (28 (link),29 (link)). For detection of RUNX3, mouse anti-human monoclonal (2B3) RUNX3 antibody (dilution 1:500; Abcam, Cambridge, CA, USA) was used. For detection of Ki-67, rabbit anti-human monoclonal (30 (link)–9 (link)) anti-Ki-67 antibody purchased from Roche Applied Science (Penzberg, Germany) was used. TMA sections were deparaffinized with toluene and rehydrated in graded alcohols. After autoclaved for 15 min at 120°C in 10 mM citrate buffer (pH 6.0) for antigen retrieval, endogenous peroxidase was inactivated with 0.3% hydrogen peroxide in methanol for 15 min. The sections were then pre-incubated with 500 µg/ml normal goat IgG dissolved in 1% BSA in PBS (pH 7.4) for 1 h, reacted with primary antibodies for 16 h, washed with 0.075% Brij 35 in PBS, and then incubated with HRP-conjugated goat anti-mouse/rabbit (RUNX3/Ki-67) in 1% BSA in PBS for 1 h. After washing with 0.075% Brij 35 in PBS, the sites of HRP were visualized with DAB and H2O2. As a negative control, some sections were reacted with normal mouse IgG instead of the specific antibodies. The stained slides were analyzed under a laser scanning microscope (LSM 5 PASCAL; Carl Zeiss AG, Oberkochen, Germany).
10
Immunohistochemistry protocol for mouse tissue
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Immunohistochemistry (IHC) was performed using Discovery Auto-Stainer with automated protocols from formalin-fixed paraffin-embedded mouse sections (Ventana Medical Systems, Inc., Tucson, AZ, USA; Roche, Mannheim, Germany). The primary antibodies used were anti-Ki67 antibody (#518–102456) from Roche (Mannheim, Germany) and anti-cleaved caspase 3 antibody (1:200 dilution; #9661) from Cell Signaling Technology.
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