Alexa fluor488 594 donkey anti rabbit mouse

The sections were incubated with 10% normal donkey serum for 1 h at room temperature in PBS containing 0.1% Triton X-100, followed by incubation with appropriate primary antibodies overnight at 4°C in the same buffer. The nuclears were stained with Hoechst 33258 (0.25 μg/ml) dye. For neurons and GAP43 detection, the following primary antibodies were used based on different targets: anti-NeuN (1:500, Millipore), anti-GAP43 (1:50, Santa Cruz, Biotechnology, Santa Cruz, CA). After primary antibody incubation, sections were washed for 4 × 10 min at room temperature, followed by incubation with Alexa Fluor594/647 donkey anti-mouse/rabbit, Alexa-Fluor488/594 donkey anti-rabbit/mouse, or Alexa-Fluor488/594 donkey anti-goat secondary antibody (1:500; Invitrogen Corporation, Carlsbad, CA, USA) for 1 h at room temperature. Sections were then washed with PBS containing 0.1% Triton X-100 for 4 × 10 min, followed by 3 × 5 min with PBS and briefly with water. All images were captured on Nikon ECLIPSE Ti microscope (Nikon, Tokyo, Japan).