Anti phosphorylatedtrkb

Immunofluorescence for brain slices was performed on fixed frozen sections as previously described (Hu et al., 2009 (link)). Twenty-four hours after SAH, rats were deeply anesthetized and transcardially perfused with phosphate-buffered solution (PBS) and 10% formalin. Rats’ brains were rapidly isolated and postfixed in 10% formalin for 24 hours and then in 30% sucrose for 3 days. Coronal brain sections (10 μm) were obtained with the help of cryostat (Leica CM3050S-3-1-1, Bannockburn, IL) and permeabilized with 0.3% Triton X-100 in PBS for 30 minutes. Sections were blocked with 5% donkey serum for 1 hour and incubated at 4 °C overnight with primary antibodies: anti-phosphorylated TrkB (Abcam, Cambridge, MA) and anti-neuronal nuclei (NeuN) (Millipore, Temecula, CA), followed by fluorescein isothiocyanate (FITC)- and Texas Red-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) for 2 hours at room temperature. The colocalization of p-TrkB with the marker of neuron was examined by fluorescent microscope (Olympus OX51, Tokyo, Japan).