Cells were labeled with antibody or the corresponding isotype control in 1%BSA/PBS for 30 mins on ice. Cells were washed and analyzed using an Accuri C6 flow cytometer (BD Bioscience); histograms were generated using FlowJo software. Mean fluorescence values were normalized to the control cell line level. Antibodies used were CD82-Alexa-647 (Biolegend, ASL-24), CD53-PE (BioLegend, HI29), and Integrin β1-Alexa-647 (BioLegend, TS2/16). For active β1 integrin expression, cells were labeled with Ligand-induced binding site (LIBS) Anti-Integrin β1 Antibody (Millipore, HUTS-4) for 30 mins. Cells were washed and labeled with goat anti-mouse Alexa-647 secondary antibody (Invitrogen).
Goat anti mouse alexa 647 secondary antibody
Manufactured by Thermo Fisher Scientific
The Goat anti-mouse Alexa-647 secondary antibody is a labeling reagent designed for immunofluorescence applications. It is a highly specific antibody that binds to mouse primary antibodies, and is conjugated to the Alexa Fluor 647 fluorescent dye, enabling the detection and visualization of target proteins or molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Asl 24, by BioLegend (2 mentions)
Cd53 pe, by BioLegend (2 mentions)
Integrin β1 alexa 647, by BioLegend (2 mentions)
Anti integrin β1 antibody, by Merck Group (2 mentions)
Cd82 alexa647, by BioLegend (2 mentions)
Accuri c6, by BD (2 mentions)
Mouse anti parvalbumin antibody, by Merck Group (1 mentions)
Hoechst, by Merck Group (1 mentions)
Orca flash 4, by Hamamatsu Photonics (1 mentions)
Axioimage m2 microscope, by Hamamatsu Photonics (1 mentions)
Goat anti mouse alexa 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Aqua poly mount, by Polysciences (1 mentions)
3 protocols using goat anti mouse alexa 647 secondary antibody
1
Flow Cytometric Analysis of Cell Surface Markers
2
Flow Cytometric Analysis of Cell Surface Markers
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Cells were labeled with antibody or the corresponding isotype control in 1%BSA/PBS for 30 mins on ice. Cells were washed and analyzed using an Accuri C6 flow cytometer (BD Bioscience); histograms were generated using FlowJo software. Mean fluorescence values were normalized to the control cell line level. Antibodies used were CD82-Alexa-647 (Biolegend, ASL-24), CD53-PE (BioLegend, HI29), and Integrin β1-Alexa-647 (BioLegend, TS2/16). For active β1 integrin expression, cells were labeled with Ligand-induced binding site (LIBS) Anti-Integrin β1 Antibody (Millipore, HUTS-4) for 30 mins. Cells were washed and labeled with goat anti-mouse Alexa-647 secondary antibody (Invitrogen).
3
Quantifying Subcellular FUS and Parvalbumin Localization
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Sections were rinsed with PBS 1X then incubated with blocking solution (8% Goat serum, 0.3% Bovine Serum Albumin, 0.3% Triton, PBS-0.02% Thimérosal) overnight at 4 °C in primary antibody: rabbit anti-FUS antibody (ProteinTech, 11570-1-AP, 1:100) and mouse anti-parvalbumin antibody (Sigma, P3088, 1:1000). After three rinses in PBS, sections were incubated for 2 h at room temperature with Hoechst (Sigma, B2261, 1/50.000) and secondary antibody: Goat anti-mouse Alexa-488 secondary antibody (Invitrogen, A11034, 1:500) and goat anti-mouse Alexa-647 secondary antibody (Invitrogen, A21245, 1:500). Finally sections were subsequently washed with PBS (3 × 10 min) and mounted in Aqua/polymount (Polysciences, 18606).
Images were acquired along the Z axis (Z stacking) using a Zeiss AxioImage.M2 microscope equipped with a Plan-Apochromat ×20/0.8 objective, high performance B/W camera (Orca Flash4, Hamamatsu) and run by the Zeiss Zen2 software. Images were quantified using the ImageJ freeware. First, the user defined ROIs corresponding to the cytoplasm and nucleus or several PV positive cells at several Z positions. Then a homemade macro was used to calculate the ratio, in the green channel, of the cytoplasm intensity divided by the nucleus one.
Images were acquired along the Z axis (Z stacking) using a Zeiss AxioImage.M2 microscope equipped with a Plan-Apochromat ×20/0.8 objective, high performance B/W camera (Orca Flash4, Hamamatsu) and run by the Zeiss Zen2 software. Images were quantified using the ImageJ freeware. First, the user defined ROIs corresponding to the cytoplasm and nucleus or several PV positive cells at several Z positions. Then a homemade macro was used to calculate the ratio, in the green channel, of the cytoplasm intensity divided by the nucleus one.
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