Mouse polyclonal anti gfap

Brain processing and immunohistochemical detection of the proliferation marker BrdU, the astrocyte and neural stem cell marker GFAP, the neural progenitor cell marker nestin, the early neuronal differentiation marker doublecortin (DCX), and the mature neuron maker NeuN were performed as previously described^{49 (link)}.
Primary antibodies used were mouse monoclonal anti-BrdU (1:100) from Dako (Hamburg, Germany) or rat monoclonal anti-BrdU (1:100) from Abcam (Cambridge, UK), mouse polyclonal anti-GFAP both from Cell Signaling (Beverly, MA, USA), goat polyclonal anti-DCX (1:500), goat polyclonal anti-nestin (1:500), and mouse monoclonal anti-NeuN (1:100) all of them from Abcam (Cambridge, UK); goat anti-ChAT, polyclonal, 1:100, Merk Millipore (Billerica, MA, USA) mouse anti-parvalbumin, monoclonal, 1:100, Merk Millipore (Billerica, Ma, USA). Secondary antibodies used were Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 594 donkey anti-mouse, Alexa Fluor 405 goat anti-mouse, Alexa Fluor 594 donkey anti-rat, Alexa Fluor 488 donkey anti-rabbit, Alexa Fluor 594 donkey anti-rabbit and Alexa Fluor 594 donkey anti-goat (all at 1:1000, from Life Tech).

The brain protein samples were extracted using RIPA buffer containing a protease inhibitor cocktail (Roche, Basel, Switzerland), and the extracted proteins were electrophoresed in a 10–12% sodium dodecyl sulfate-polyacrylamide gel (Willget, China). Thereafter, the separated proteins were transferred onto polyvinylidene difluoride membranes; the membranes were then probed overnight with the following primary antibodies: mouse polyclonal anti-GFAP (1:800; cat# 3670; Cell Signaling Technology, MA, United States), rabbit polyclonal anti-MAP2 (1:800; cat# 4542; Cell Signaling Technology) and mouse polyclonal anti-GAPDH (1:2,000, sc-365062, Santa Cruz Biotechnology, TX, USA). On the next day, after washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The proteins were then visualized using an enhanced chemiluminescent substrate (ECL; Thermo Fisher Scientific, IL, United States).