The Fixation/Permeabilization solution and Perm/WashTM Buffer (BD Biosciences, San Jose, CA, USA) were used. The anti-Sox2 antibody (S9072, Sigma-Aldrich) and the secondary Alexa Fluor® 647-conjugated antibody (Thermo Fisher Scientific, Waltham, MA, USA) were diluted 1:200 and 1:500, respectively. The FACS Canto instrument and the FACS Diva and FlowJo software version 6.1.1 (BD Biosciences) were used for the analysis.
Alexa fluor 647 conjugated antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 647-conjugated antibody is a fluorescent-labeled antibody product manufactured by Thermo Fisher Scientific. It is designed for use in various immunological and cell biology applications that require labeling and detection of target proteins or cells.
Automatically generated - may contain errors
Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Anti phospho smad1 5 8 antibody, by Cell Signaling Technology (2 mentions)
Evos fl imaging system, by Thermo Fisher Scientific (2 mentions)
Blockaid blocking solution, by Thermo Fisher Scientific (2 mentions)
Rabbit anti phospho h2afx, by Merck Group (2 mentions)
Alexa fluor 488 conjugated antibody, by Thermo Fisher Scientific (2 mentions)
β actin antibody, by Santa Cruz Biotechnology (2 mentions)
γh2ax, by Cell Signaling Technology (2 mentions)
Sox9 antibody, by Merck Group (2 mentions)
Sycp3, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Zhongshan Golden Bridge Biotechnology (2 mentions)
Sodium citrate, by Thermo Fisher Scientific (2 mentions)
D9663, by Merck Group (2 mentions)
Cm1950, by Leica (2 mentions)
Anti β amyloid antibody, by Cell Signaling Technology (2 mentions)
G5516, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Perm wash buffer, by BD (1 mentions)
26 protocols using alexa fluor 647 conjugated antibody
1
Sox2 Expression Analysis by Flow Cytometry
2
Phospho-SMAD1/5/8 Immunostaining
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Embryos were penetrated in 0.1% Triton X-100 in PBS for 1 h at RT. Embryos were blocked in blocking buffer ( 4% BSA(Millipore Sigma, #126,615), 1% DMSO, 0.1% Triton X-100 in PBS) overnight at 4 °C, and then stained with anti-phosphoSmad1/5/8 antibody (Cell Signaling Technology, #9511) at 1:100 diluted in blocking buffer overnight at 4 °C. Then embryos were detected by goat anti-rabbit cross-adsorbed Alexa Fluor 647-conjugated antibody at 1:500 dilution with DAPI overnight at 4 °C (Thermo Fisher Scientific, #A21244).
3
Multiparametric Flow Cytometric Analysis of Immune Cell Subsets
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For surface marker detection, cells were stained with anti-CD45, anti-CD4, anti-CD8, anti-B220, anti-CD11b, anti-F4/80, anti-Ly-6C, and anti-Ly-6G antibodies. For Th1 and Th17 cell analysis, cells were stimulated with phorbol-12-myristate-13-acetate (50 ng/mL) and ionomycin (500 ng/mL; Sigma) in the presence of brefeldin A (BD Bioscience, San Diego, CA, USA) for 5 h. Then, the cells were incubated with an anti-CD4 antibody, permeabilized with permeabilization buffer (Thermo Fisher Scientific), and incubated with anti-IFN-γ and anti-IL-17A antibodies. Treg cells were detected using a mouse Treg cell staining kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. For determining intracellular levels of mTOR, CD4+ T cells were permeabilized with permeabilization buffer and stained with anti-mTOR antibody (Cell Signaling Technology, Danvers, MA, USA) for 60 min, followed by the secondary antibody, a Alexa-Fluor 647-conjugated antibody (Thermo Fisher Scientific) for 30 min in the dark at 4°C. For the glucose uptake assay, prestained cells were incubated with 10 μM 2-NBDG (Thermo Fisher Scientific) for 30 min, followed by flow cytometric detection of fluorescence produced by the cells. All stained cells were analyzed by flow cytometry on a BD FACSCalibur (BD Bioscience).
4
Immunostaining and Confocal Imaging of Embryos
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Embryos were fixed overnight in 4% paraformaldehyde at 4°C, blocked in NCS-PBST (10% fetal bovine serum, 1% DMSO, 0.1% Tween 20 in PBS), and probed overnight with a 1:100 dilution of anti-phosphoSmad1/5/8 antibody (Cell Signaling Technology, #9511, discontinued), followed by a 1:500 dilution of goat anti-rabbit Alexa Fluor 647-conjugated antibody (Thermo Fisher Scientific, Rockford, IL; Cat# A-21244, RRID:AB_2535812 ). Embryos were mounted in BABB (benzyl alcohol (Sigma B-1042) and benzyl benzoate (Sigma B-6630), 1:2 ratio) and scanned using a Zeiss LSM 710 confocal microscope with a LD LCI Plan-Achromat 25x/0.8 Imm Corr DIC M27 multi-immersion lens. The oil-immersion setting was used to reduce Mie scattering distortion, spherical aberrations, and chromatic aberrations by minimizing refractive index (R.I.) mismatch between the lens oil (R.I. = 1.518), the coverslip, BABB (R.I.≈1.56), and the light scattering particles in the embryo (R.I.≈1.56). Fluorophore bleaching was greatly reduced by precise embryo orientation, reducing sample thickness, and by high scan speeds using a Zeiss LSM 710 confocal microscope. Nuclei were visualized with Sytox Orange (Molecular Probes) or Sytox Green (Molecular Probes).
5
Quantifying DNA Damage with γ-H2AX Immunofluorescence
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H2Ax is a core histone protein found in the nucleosome, and the phosphorylation of H2Ax is a marker of DNA damage caused by ROS. Variant histone H2AX (γ-H2AX) is a marker of DNA double-strand breaks (Rothkamm et al., 2015 (link)). Exposed cells (1 week with 10 µM of compound) were plated in a 12-well cell culture treated plate at 1x105 cells/well. Following 24 h incubation at 37°C in media containing 10 µM of compound, cells were fixed with a cold 1:1 solution of methanol and acetone for 20 min at -20°C. The cells were subsequently washed with PBS and blocked for 2 h at room temperature with 1 ml of BlockAid™ Blocking Solution (Thermo Scientific). Each well was stained with a 1:1,000 dilution of rabbit anti-phospho-H2AFX (Millipore Sigma, St. Louis, MO) in PBS overnight at 4°C with gently rocking. Following multiple washing steps with PBS, cells were incubated with 5 µg/ml of anti-rabbit Alexa Fluor 647-conjugated antibody (ThermoFisher) for 1 h at room temperature. Cells were subsequently washed with PBS and stained with 1 µg/ml of Hoechst 33342 (ThermoFisher) for 5 min at room temperature. Fluorescent images were acquired on an Evos FL Imaging System (ThermoFisher).
6
Zebrafish Embryo TUNEL Assay
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The zebrafish embryos were fixed using 4% paraformaldehyde at 4 °C and dehydrated using methanol, at − 20 °C. The dehydrated samples were rehydrated using PBT and permeabilized using PBS with 0.5% Triton X-100 and 1% DMSO. After permeabilization, the samples were post-fixed using ethanol:acetic acid (2:1) at − 20 °C, and blocked in PBS containing 5% BSA at 4 °C. The samples were washed using PBT and blocked with biotin using a Streptavidin/Biotin Blocking Kit (Vector Laboratories, Newark, CA, USA). After blocking, the samples were incubated in equilibration buffer (PBS with 1 × TdT reaction buffer and 1 × CoCl2; Roche), which was then replaced with TdT reaction solution (equilibration buffer with 600 units of Terminal Transferase and Biotin-16 UTP; Roche). For TUNEL signal development, the samples were incubated in PBT with streptavidin, Alexa Fluor™ 647-conjugated antibody (1:5000, Thermo Fisher Scientific, Waltham, MA, USA), and mounted in VECTASHIELD® Antifade Mounting Medium with DAPI (Vector Laboratories).
7
Quantifying DNA Damage via γ-H2AX Assay
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H2Ax is a core histone protein found in the nucleosome, and the phosphorylation of H2Ax is a marker of DNA damage caused by ROS. Variant histone H2AX (γ-H2AX) is a marker of DNA double-strand breaks 18 . Exposed cells (1 week with 10 µM of compound) were plated in a 12-well cell culture treated plate at 1x10 5 cells/well. Following 24 hours incubation at 37°C in media containing 10 µM of compound, cells were fixed with a cold 1:1 solution of methanol and acetone for 20 min at -20°C. The cells were subsequently washed with PBS and blocked for 2 hours at room temperature with 1 mL of BlockAid™ Blocking Solution (Thermo Scientific). Each well was stained with a 1:1000 dilution of rabbit anti-phospho-H2AFX (Millipore Sigma, St. Louis, MO) in PBS overnight at 4°C with gently rocking.
Following multiple washing steps with PBS, cells were incubated with 5 µg/mL of anti-rabbit Alexa Fluor 647-conjugated antibody (ThermoFisher) for 1 hour at room temperature. Cells were subsequently washed with PBS and stained with 1 µg/mL of Hoechst 33342 (ThermoFisher) for 5 min at room temperature. Fluorescent images were acquired on an Evos FL Imaging System (ThermoFisher).
Following multiple washing steps with PBS, cells were incubated with 5 µg/mL of anti-rabbit Alexa Fluor 647-conjugated antibody (ThermoFisher) for 1 hour at room temperature. Cells were subsequently washed with PBS and stained with 1 µg/mL of Hoechst 33342 (ThermoFisher) for 5 min at room temperature. Fluorescent images were acquired on an Evos FL Imaging System (ThermoFisher).
8
Immunohistochemical Analysis of Aortic eNOS and Akt
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Frozen sections of the aortas (7 μm thick) were fixed with cold 100% methanol and incubated for 2 h at room temperature in blocking buffer (5% of albumin in PBS). Tissue sections were then incubated overnight (4°C) with a mouse monoclonal antibody against the phosphorylated-(Ser 1179)-eNOS (1 : 50, Santa Cruz Biotechnology) or a rabbit polyclonal antibody against phosphorylated-(Ser473)-Akt (p-Akt) protein (1/500, Cell Signaling). Three washes were followed by incubation (1 h, at room temperature, in the dark) with the secondary mouse or rabbit fluorescent Alexa fluor-647-conjugated antibody (1 : 500, Molecular Probes). A Nikon A1-RS inverted laser scanning confocal microscope was used for the optical sectioning of the tissue. Digital image recording was performed using the NIS element software. Images were analysed and processed by Fiji software.
9
Quantifying DNA Damage and Mitochondrial Oxidative Stress
10
Intracellular CagA Detection in AGS Cells
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AGS cells were seeded in μ-slide eight well-chambers (Ibidi) at 3 × 104 cells/ml and incubated for 24 h at 37°C in 5% CO2. Cells were then serum-starved overnight prior to co-culture with H. pylori for 6 h. Cells were subsequently fixed using 4% paraformaldehyde and their nuclei stained with DAPI (diluted 1:1,000 in PBS; Molecular Probes). Extracellular CagA was detected using a rabbit anti-CagA (b-300) antibody (1:100; sc-25766, Santa-Cruz Biotechnology), followed by incubation with an anti-rabbit Alexa Fluor 488 conjugated antibody (1:1,000 dilution; Molecular Probes). Cells were then re-fixed and permeabilized using 1% Triton-X. Intracellular CagA was probed using the same rabbit anti-CagA antibody, followed by incubation with an anti-rabbit Alexa Fluor 647 conjugated antibody (1:1,000 dilution; Molecular Probes). Cells were imaged with the same background intensity settings on a Deltavision API wide-field microscope (60 x objective). Image analysis was performed using ImageJ, where relative intensities of A647/A488 were quantified for five cells per field viewed (five fields viewed per sample; n = 2 experiments).
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