NSCs were fixed in 4% paraformaldehyde for 24 h at room temperature and then permeabilized by using 0.2% Triton-X for 1 h at room temperature. Following blocking with 10% goat serum (Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at room temperature, cells were treated with rabbit anti-Ki67 (cat. no. 612254; 1:250; BD Biosciences, Franklin Lakes, NJ, USA) and anti-thyroid hormone receptor interactor 6 (TRIP6; cat. no. 612254; 1:250; BD Bioscience, Franklin Lakes, NJ, USA) antibodies at 4°C overnight, followed by incubation with DyLight-549 goat anti-rabbit antibody (cat. no. 012-500-003; 1:500; Jackson ImmunoResearch, West Grove, PA) according to the manufacturer's instructions. Sections were washed with PBS and incubated with 0.5 µg/ml 4,6-Diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA, USA) solution for 30 min in the dark at room temperature; DAPI was used for nuclear staining in order to maintain an analogous background. Cells were imaged using an inverted fluorescence microscope (Leica DMI3000; Leica Microsystems, Inc.). The labelled nuclei and fluorescence intensity were analysed in an average of 5 high-powered fields (magnification, ×200) using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MD, USA).
Rabbit anti ki67
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
Rabbit anti-Ki67 is a primary antibody that binds to the Ki-67 protein, a well-established marker of cellular proliferation. The Ki-67 protein is expressed during active phases of the cell cycle (G1, S, G2, and mitosis) but is absent in resting cells (G0). This antibody can be used to detect and quantify the proliferative state of cells in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Rabbit anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Rat anti brdu, by Abcam (3 mentions)
Biotinylated donkey anti rabbit igg, by Vector Laboratories (3 mentions)
Avidin horseradish peroxidase conjugates, by Vector Laboratories (3 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Mouse anti brdu, by Thermo Fisher Scientific (2 mentions)
Citrate buffer, by Merck Group (2 mentions)
Mouse anti tnnt2, by Thermo Fisher Scientific (2 mentions)
Rabbit anti tnni3, by Santa Cruz Biotechnology (2 mentions)
Lsm 510 meta inverted confocal microscope, by Zeiss (2 mentions)
Rabbit anti phospho smad2, by Merck Group (2 mentions)
Rabbit anti prdm16, by Abcam (2 mentions)
Rabbit anti phospho histone h3, by Cell Signaling Technology (2 mentions)
Rabbit anti tgf β1, by Abcam (2 mentions)
Eclipse 80i fluorescent microscope, by Nikon (2 mentions)
Mouse anti sarcomeric alpha actinin, by Merck Group (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Relevant secondary antibodies, by Abcam (2 mentions)
Tmr red, by Roche (2 mentions)
45 protocols using rabbit anti ki67
1
Immunofluorescent Staining of Neural Stem Cells
2
Immunohistochemistry and Immunofluorescence of Cell Markers
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Sections of paraffin-embedded neutralized buffered formalin fixed tissue were used for Ni-DAB (FoxM1) or DAB (other antibodies) colorimetric immunohistochemistry (IHC), while paraformaldehyde fixed frozen tissue sections or cells grown on coverslips were subjected to immunofluorescence (IF). Antibodies include rabbit anti-FoxM1 (1:1000 for IHC or 1:500 for IF; Santa Cruz, cat# sc-502), rabbit anti-cyclin D3 (1:500; Santa Cruz), rabbit anti-Ki67 (1:500; Thermo), rat anti-BrdU (1:200; Abcam), rat anti-phosphorylated histone H3 (Ser 10) (1: 1000; Millipore), rabbit anti-PR (1:300; Santa Cruz), rabbit anti-ERα (1:300; Santa Cruz), and rabbit anti-cleaved caspase 3 (1:300 for IF; Santa Cruz). Counting of immunostaining-positive cells were determined using the Image J program available at http://imagej.nih.gov/ij (NIH, USA), and the analyses were based on examination of serial sections for at least 5–6 IS samples collected from 3–5 different mice for each group.
3
Immunohistochemical Analysis of Intestinal Markers
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Tissue sections were immunostained as previously described [28 (link)]. Primary antibodies included rabbit anti-Ki67 (Thermo Fisher Scientific, Inc., Fremont, CA; Cat. No. RM-9106-S1) (1:200), mouse anti-BrdU (Thermo; Cat. No. MS-1058-PO) (1:250), goat anti-cryptdin related sequence 4C (CRS4C) (gift from Dr. A. J. Ouellette, University of Southern California, Los Angeles, CA) [32 (link)] (1:2000), and rabbit anti-MUC2 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA; Cat. No. sc15334) (1:100). Secondary antibodies included biotinylated donkey anti-rabbit IgG, donkey anti-goat IgG, and donkey anti-mouse IgG (all from Vector Labs, Burlingame, CA). Biotinylated antibodies were linked to avidin-horseradish peroxidase conjugates (Vector Labs), visualized using 3,3′-diamino benzidine (Sigma) for 2 to 5 min, and lightly counterstained with hematoxylin.
4
Tissue Processing for Protein and Histology
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Every tumor was cut in half with a scalpel blade; half was taken for protein analysis and half for immunohistochemistry and histology. For protein analysis, tumors were homogenized by a polytron homogenizer in lysis buffer, immunoblot and immunopercipitations were done as described above. For immunostaining tumors were post-fixed for 3 hours in cold 4% PFA followed by 20% sucrose in PBS overnight at 40C. Cryostat sections (20 μm) were cut and stained using standard immunohistochemistry as described above. Primary antibodies: rabbit anti-Ki67 (1:300, Thermo Scientific), rabbit anti-active caspase 3 (1:1000, R&D Systems). Standard protocol was used for H&E staining.
5
Immunohistochemical Analysis of Apoptosis and Cell Proliferation
6
Antibody Staining Panel for Neural Development
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Primary antibodies used included rabbit anti-Kif2a (1: 1000, Abcam), rabbit anti-Tbr2 (1:500, Abcam), rabbit anti-Pax6 (1:200, Covance), mouse anti-beta III tubulin (TU20) (1:500, Abcam), mouse anti-nestin (1:500, Abcam), mouse anti-α tubulin (1:5000, CST), rat anti-Brdu (1:500, Abcam), rabbit and chicken anti-green fluorescent protein (1:500, GFP) (Invitrogen and Aves, respectively), rabbit anti-Ki67 (1:500, Thermo), rabbit anti-PH3 (1:300, Millipore), rabbit anti-cleaved caspase3 (1:300, Cell signaling), rabbit anti-AKT (1:500, CST), rabbit anti-p-AKT (1:500, CST), rabbit anti-β-catenin (1:200, Santa Cruz), mouse anti-GSK3β (1:200, Santa Cruz), and mouse anti-p-GSK3β (1:200, Santa Cruz). All the corresponding conjugated secondary antibodies were purchased from Invitrogen. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Roche).
7
Immunofluorescence Analysis of Cardiac Development
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Mouse embryos and neonates were collected, fixed in 4% PFA, embedded in OCT, and frozen in dry iced hexane. Frozen sections were blocked with 5% goat serum, stained with mouse anti-TNNT2 (Thermo Scientific), mouse anti-sarcomeric alpha-actinin (Sigma Aldrich), rabbit anti-TNNI3 (Santa Cruz), rabbit anti-TGFβ1 (Abcam), rabbit anti-Ki67 (Thermo Scientific), rabbit anti-phospho-Histone H3 (Cell Signaling Technology), and Hoechst 33342, and visualized using Alexa Fluor-conjugated secondary antibodies (Life Technologies). Image acquisition was performed on a Zeiss LSM510 Meta inverted confocal microscope (Carl Zeiss) or an Eclipse 80i fluorescent microscope (Nikon). Transgenes were detected by PCR from yolk sac DNA of embryos or tail DNA of neonatal pups. For human heart tissues, formalin-fixed paraffin embedded tissue-sections were rehydrated and autoclaved with Citrate Buffer, pH 6.0 (Sigma-Aldrich) and stained with rabbit anti-phospho-Smad2 (EMD Millipore), rabbit anti-PRDM16 (Abcam), mouse anti-sarcomeric alpha-actinin (Sigma Aldrich) and Hoechst 33342, and visualized using Alexa Fluor-conjugated secondary antibodies (Life Technologies). Image acquisition was performed on an Eclipse 80i fluorescent microscope (Nikon). A detailed list of antibodies used is shown in Supplementary Table 10 .
8
Peroxidase Immunohistochemistry for BrdU and Ki67
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Peroxidase immunohistochemistry was performed essentially as described previously.43 (link) Briefly, floating sections were incubated in hydrogen peroxide (1% in PBS) to destroy endogenous peroxidase activity; underwent antigen unmasking in 1 m HCL for 35 min at 37 °C (for BrdU only); were incubated in blocking buffer consisting of 0.2% Triton X (Sigma-Aldrich) and 5% v/v normal donkey serum (Merck-Millipore, Kilsyth, VIC, Australia) in 100 mm PBS for 1 h; were incubated in either sheep anti-BrdU (Exalpha Biologicals, Watertown, MA, USA) diluted 1/1000 or rabbit anti-Ki67 (Thermo Fisher Scientific Australia, Scoresby, VIC, Australia) diluted 1/200 in blocking buffer overnight at 20 °C; were incubated with biotinylated rabbit anti-sheep (Vector Laboratories, Burlingame, CA, USA) or biotinylated goat anti-rabbit Ki67 (Vector Laboratories) diluted at 1/500 for 2 h; incubated with Vectastain ABC solution (1:100, Vector Laboratories) for 1 h; developed with diaminobenzidine liquid chromogen kit (1:50, Dako Australia, North Sydney, NSW, Australia); were mounted on slides and then cover-slipped with DPX. Unless otherwise stated, each step occurred at room temperature and was followed by thorough washing with 100 mm PBS.
9
Immunofluorescence Analysis of Cardiac Development
10
Immunohistochemical Analysis of Ki67 Expression
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