We performed experiments with cell cultures to validate the viral-mediated manipulation of NMDAR expression. Primary neuronal cultures were prepared as previously described [29 (link)]. Briefly, cortical neurons at E18 from pregnant female rats were dissected, dissociated and plated in wells (~1 million cells/well) and cultured in standard media. Viruses were added at DIV5 stage. To validate GluN1 overexpression, we used the following viruses: AAV1-CMV-Cre-GFP (1 × 1013 GC/ml titer, 400 nL) and LV-Syn-DIO-GluN1–2A-tdTomato (2 × 109 TU/ml titer, 2 μL). Using these viruses, we made the four well conditions: GluN1, no Cre; GluN1, Cre; no GluN1, Cre; no GluN1, no Cre (no viruses added). After at least 21 days of culture, we performed Western blot for GluN1 (#5704S, Cell Signaling, 1:500, rabbit monoclonal antibody) and beta-actin (#4967S, Cell Signaling, 1:1000, rabbit polyclonal antibody). To validate GluN2B overexpression, we used identical procedures except that we used LV-Syn-DIO-mTagBFP2–2A-GluN2B (1 × 109 TU/ml titer, 2 μL) and GluN2B antibody (#4207S, Cell Signaling, 1:1000, rabbit polyclonal antibody). Bands were quantified using Bio-Rad Image Lab software and raw band intensity values were normalized to beta-actin levels.
Glun2b antibody
Manufactured by Cell Signaling Technology
The GluN2B antibody is a research tool used to detect the GluN2B subunit of the NMDA receptor in various sample types. It can be used in techniques such as Western blotting, immunohistochemistry, and immunocytochemistry to study the expression and localization of the GluN2B subunit.
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2 protocols using glun2b antibody
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Viral Manipulation of NMDAR Expression
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Viral Manipulation of NMDAR Expression
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We performed experiments with cell cultures to validate the viral-mediated manipulation of NMDAR expression. Primary neuronal cultures were prepared as previously described [29 (link)]. Briefly, cortical neurons at E18 from pregnant female rats were dissected, dissociated and plated in wells (~1 million cells/well) and cultured in standard media. Viruses were added at DIV5 stage. To validate GluN1 overexpression, we used the following viruses: AAV1-CMV-Cre-GFP (1 × 1013 GC/ml titer, 400 nL) and LV-Syn-DIO-GluN1–2A-tdTomato (2 × 109 TU/ml titer, 2 μL). Using these viruses, we made the four well conditions: GluN1, no Cre; GluN1, Cre; no GluN1, Cre; no GluN1, no Cre (no viruses added). After at least 21 days of culture, we performed Western blot for GluN1 (#5704S, Cell Signaling, 1:500, rabbit monoclonal antibody) and beta-actin (#4967S, Cell Signaling, 1:1000, rabbit polyclonal antibody). To validate GluN2B overexpression, we used identical procedures except that we used LV-Syn-DIO-mTagBFP2–2A-GluN2B (1 × 109 TU/ml titer, 2 μL) and GluN2B antibody (#4207S, Cell Signaling, 1:1000, rabbit polyclonal antibody). Bands were quantified using Bio-Rad Image Lab software and raw band intensity values were normalized to beta-actin levels.
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