Microtubule associated protein 2 map 2

The cells were xed with 4% paraformaldehyde in PBS, permeabilized, and blocked with 0.3 % Triton-X 100 plus and 5 % normal bovine serum in PBS. The cultures were then incubated with combinations of the primary antibodies against the following targets: microtubule-associated protein 2 (MAP-2; a neuronal marker; Santa Cruz) and synaptophysin (is an integral membrane protein localized to synaptic vesicles; Santa Cruz) and diluted in 5% BSA overnight at 4 °C. Then, cortical neurons were incubated with the appropriate secondary antibodies (Alexa Fluor 568-conjugated goat anti-mouse IgG or Alexa Fluor 488conjugated goat anti-mouse) for 1 h at room temperature. The neurons were also stained with DAPI (Sigma-Aldrich) as a nuclear marker. Their digital images were captured with a 20x objective (N.A. 0.75) by a uorescence microscope system (Carl Zeiss Microscopy GmbH, Jena, Germany) with Zen Software (Carl Zeiss). Synaptic density was measured as a synaptophysin-positive area. At least 10 elds obtained from different experiments were conducted in triplicate from 3 cultures, were captured and measured for each condition. Values obtained for each eld (0.15 mm 2 ) were pooled to obtained mean values for each culture.