Donkey anti rat alexa 568

The maturation and activation marker expression of newborn neurons were assessed using triple fluorescence labeling for BrdU, NeuN and c-fos (Deng et al., 2009). One of the six serial sections was treated with 2 M HCl for 30 minutes at 37°C and subsequently incubated simultaneously in three primary antibodies, diluted in PBS containing 0.5% Triton-X and 3% donkey serum, for 48 hours at 4°C. Primary antibodies included rat anti-BrdU polyclonal antibody (1:1,000; Accurate Chemical & Scientific Corporation Company, Philadelphia, PA, USA), goat anti-NeuN antibody (1:400; Cell Signaling Technology Inc.) and rabbit anti-c-fos polyclonal antibody (1:500; Cell Signaling Technology Inc.). After incubation of primary antibodies, donkey anti-rat Alexa 568, donkey anti-goat Alexa 647 and donkey anti-rabbit Alexa 488 antibodies (1:250; Invitrogen Life Technologies, Carlsbad, CA, USA) were added onto the slides for 90 minutes at room temperature. After three rinses, the slides were mounted with florescence mounting media (DAKO, Carpinteria, CA, USA) and coverslipped.