The REST gene was overexpessed in NP cells by lentiviral transduction of a construct encoding the ubiquitin C promoter and REST or GFP as a control, as previously described (Lu et al., 2014 (link)). DCX gene expression was measured 1 and 2 weeks after infection, while quantification of all other genes and was performed after 1 week. Cells were labeled for nestin, DCX, and DAPI according to the above procedure with secondary antibodies of α-mouse IgG Alexafluor-647 (A21235, Life Technologies) and α-rabbit DyLight 594 (Abcam). DCX-positive cells were quantified 1 week after infection using MetaMorph (v7.7.0.0) software with consistent inclusion criteria. Data is the mean from four different experiments with greater than 3 fields scored per experiment. REST knockdown was performed using either of two distinct shRNAs by lentiviral transduction as previously described (Lu et al., 2014 (link)). An shRNA with a scrambled sequence was used as a control. DCX expression levels were measured 1 week after infection. REST overexpression was confirmed by qRT-PCR and REST knockdown by western blotting.
α mouse igg alexafluor 647
Manufactured by Thermo Fisher Scientific
The α-mouse IgG Alexafluor-647 is a fluorescent-labeled secondary antibody that recognizes mouse immunoglobulin G (IgG) antibodies. The Alexa Fluor 647 dye is conjugated to the secondary antibody, providing a far-red fluorescent signal that can be detected using appropriate instrumentation.
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2 protocols using α mouse igg alexafluor 647
1
Overexpression and Knockdown of REST in Neural Progenitor Cells
2
Overexpression and Knockdown of REST in Neural Progenitor Cells
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The REST gene was overexpessed in NP cells by lentiviral transduction of a construct encoding the ubiquitin C promoter and REST or GFP as a control, as previously described (Lu et al., 2014 (link)). DCX gene expression was measured 1 and 2 weeks after infection, while quantification of all other genes and was performed after 1 week. Cells were labeled for nestin, DCX, and DAPI according to the above procedure with secondary antibodies of α-mouse IgG Alexafluor-647 (A21235, Life Technologies) and α-rabbit DyLight 594 (Abcam). DCX-positive cells were quantified 1 week after infection using MetaMorph (v7.7.0.0) software with consistent inclusion criteria. Data is the mean from four different experiments with greater than 3 fields scored per experiment. REST knockdown was performed using either of two distinct shRNAs by lentiviral transduction as previously described (Lu et al., 2014 (link)). An shRNA with a scrambled sequence was used as a control. DCX expression levels were measured 1 week after infection. REST overexpression was confirmed by qRT-PCR and REST knockdown by western blotting.
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