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3 protocols using mouse monoclonal anti gapdh mab374

1

Western Blot Analysis of Protein Extracts

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The cells were lysed 48 h posttransfection in radioimmunoprecipitation assay buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.2% sodium dodecyl sulfate, 50 mM Tris-HCl, 1× Protease inhibitor cocktail Complete (Roche), and 1 mM DTT). The protein concentrations were measured with Pierce BCA Protein Assay Kit (Thermo Scientific).
Equal amounts of protein were separated in 8 to 10% SDS-polyacrylamide gel and transferred to Immobilon-P polyvinylidene fluoride membrane (Millipore). The membrane was blocked with 5% skimmed milk in PBS-0.1% Tween 20 (PBST) (Sigma-Aldrich) at room temperature and incubated with primary and secondary antibody solutions in 2% skimmed milk in PBST. The antibodies were used in the following dilutions: rabbit polyclonal anti-TCF4 (CeMines) 1:1000, mouse monoclonal anti-E2 (5E11, Icosagen,) 1:5000, mouse monoclonal anti-GAPDH (MAB374, Millipore) 1:4000, horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG (Thermo Scientific) 1:5000. For chemiluminescent reaction, SuperSignal West Femto Maximum Sensitivity Substrate Kit (Thermo Scientific) was used, and the reaction was visualized with ImageQuant LAS4000 camera system (GE Healthcare).
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2

Immunoblot Analysis of Neural Markers

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For western blot analysis, the following primary antibodies were used: mouse monoclonal anti-GFAP (ab4648; Abcam, Cambridge, UK), mouse monoclonal anti-Tuj1 (MMS435; Covance, Princeton, NJ, USA), mouse monoclonal anti-P53 (2524; Cell Signaling Technology Inc., Boston, MA, USA), rabbit monoclonal anti-p-CREB (9198; Cell Signaling Technology Inc.), rabbit monoclonal anti-p-PKA (5661; Cell Signaling Technology Inc.) and mouse monoclonal anti-GAPDH (MAB374; Millipore, Billerica, MA, USA).
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3

Immunohistochemical Staining Protocol

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The following primary antibodies were used: goat polyclonal anti-AQP4 (Santa Cruz, CA, USA); goat polyclonal anti-CD31 (Santa Cruz, CA, USA); rabbit polyclonal anti-GFAP (Sigma-Aldrich, Milan, Italy); mouse monoclonal anti-GFAP (clone G-A-5, Millipore, Merck KGaA, Darmstadt, Germany); mouse monoclonal anti-GAPDH (MAB 374, Millipore, Merck KGaA, Darmstadt, Germany); mouse monoclonal anti-Glutamine Synthetase (MAB 302, Millipore, Merck); goat polyclonal anti-actin (Santa Cruz, CA, USA). The following secondary antibodies (all from Invitrogen, Milan, Italy) were used for immunofluorescence: donkey anti-goat Alexa Fluor488; donkey anti-mouse Alexa Fluor488; donkey anti-rabbit Alexa Fluor594; donkey anti-goat Alexa Fluor594; donkey anti-rabbit Alexa Fluor647. The following secondary antibodies (all from Santa Cruz, CA, USA) were used for Western blot:
goat anti-mouse IgG-horseradish peroxidase (HRP); goat anti-rabbit IgG-HRP; donkey anti-goat IgG-HRP.
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