Donkey anti goat alexa fluor 594 secondary antibody

All sections received CB-HRP immunofluorescent staining (rabbit anti-CB primary antibody diluted in 1:600, Abcam; donkey anti-rabbit Alexa Fluor 488 secondary antibody diluted in 1:200, Life Technologies). Then, the sections were mounted in sequence on the slides, counterstained by DAPI, and coverslipped. The sections of different positions were captured at uniform parameter using a fluorescence microscope (Leica DM6, Germany). The number of positive neurons was counted by the Image-Pro Plus 7.0 software. Part of the sections received CB-HRP/serotonin immunofluorescent double labeling to confirm the neurotransmitter (rabbit anti-CB primary antibody diluted in 1:600, Abcam; goat anti-serotonin primary antibody diluted in 1:600, Abcam; donkey anti-rabbit Alexa Fluor 488 secondary antibody diluted in 1:200, Life Technologies; donkey anti-goat Alexa Fluor 594 secondary antibody diluted in 1:200, Life Technologies).

After fixation, the brain and the spinal cord were immersed in a 30% sucrose solution until it sank to the bottom. Serial sections (50 μm thick) of the brain and the spinal cord were prepared using a freezing microtome (Leica, Germany). The sections were collected in phosphate buffer saline (PBS) for immunofluorescence staining. The sections were incubated with anti-CB primary antibody (1:600 dilution, List Biological Labs) at 4°C for overnight, followed by incubation with donkey anti-goat Alexa Fluor 594 secondary antibody (1:200 dilution, Life Technologies). Some of the sections were retained for CB-HRP/NeuN double immunofluorescence staining. The CB-HRP stained sections were incubated with anti-NeuN primary antibody (1:50 dilution, Cell Signaling Technology) at 4°C overnight, followed by incubation with donkey anti-rabbit Alexa Fluor 488 secondary antibody (1:200 dilution, Life Technologies). The sections were then mounted in sequence on the slides and counterstained with DAPI before covering with coverslips. The sections were observed and images were captured on a confocal microscope (Zeiss, Germany) using uniform parameters. After capturing the images, some sections were reused for Nissl staining to observe the neural morphology and Nissl body distributions.