Fluorescent secondary antibody

The skins were fixed in 4% paraformaldehyde and 20-μm cryosections were prepared. The sections were incubated with a blocking buffer (1 × PBS/5% normal goat serum/0.3% triton X-100) for 1 h. The sections were incubated with the following primary antibodies at 4 °C overnight in PBS: pAkt (Cell Signaling Technology), pp38 (Cell Signaling Technology), and pNF-κB (Cell Signaling Technology). After washing with PBS, the sections were incubated with fluorescent secondary antibody (Vector Laboratories Inc.) for 2 h in the dark. The slides were mounted with mounting medium (Vector Laboratories Inc.). An optical microscope (Axio Vision LSM 510, Carl Zeiss Inc.) was used for measuring the fluorescence intensity.

Frozen sections (30-μm thickness) were incubated with a blocking buffer (1× PBS/5% normal goat serum/0.3% Triton X-100) for 1 h. Sections were incubated with primary antibodies overnight in an antibody dilution buffer (1× PBS/1% BSA/0.3% Triton X-100) at 4 °C. The antibodies were as follows: BrdU (Cat. OBT0030G, AbD Serotec, Oxford, UK), NeuN (Cat. MAB377; ABN78, Millipore Corporation, Billerica, MA, USA), Syn1 (Cat. ab64581, Abcam, Cambridge, UK), TH (Cat. AB152, Millipore Corporation), and pCREB (Cat. sc-7978-R, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The sections were incubated with a fluorescent secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA) for 2 h and 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen Corporation, Carlsbad, CA, USA) for 30 min in the dark, respectively. After slide mounting with a mounting medium (Vector Laboratories, Inc.), images were captured using a fluorescence microscope (Carl Zeiss, Inc.).

Mice anesthetized with isoflurane received intracardial perfusion with saline and then 4 % paraformaldehyde in PBS. Brains were removed, post-fixed in the same fixative for 4 h at 4 °C, and immersed in 30 % sucrose for 48 h at 4 °C for cryoprotection. Frozen 20 μm-thick sections were incubated for blocking with a blocking buffer (1X PBS/5 % normal serum/0.3 % Triton X-100) for 1 h at room temperature. The sections were incubated with the following primary antibodies to NeuN (Millipore Corporation), mature BDNF (Abcam), and pCREB (Cell Signaling) overnight in PBS at 4 °C. After washes with PBS, the sections were incubated with the fluorescent secondary antibody (Vector Laboratories, Inc., Burlingame, CA, USA) at room temperature in the dark, respectively, and then washed three times with PBS. Subsequently, slides were mounted in the mounting medium (Vector Laboratories, Inc.) and captured using a fluorescence microscope.