Ab75040

For immunofluorescence analysis, 25 μm serial cryosections were obtained using a CM3050 cryostat (Leica Microsystems, Wetzlar, Germany). After incubation in a blocking buffer (1 × PBS/1% bovine serum albumin/0.3% Triton X-100) for 1 hr at room temperature, sections were incubated with the following primary antibodies overnight in PBS at 4°C: mBDNF (ab75040, Abcam), pTrkB (ab131483, Abcam), TH (sc25269, Santa Cruz Biotechnology, Inc), DAT (sc32259, Santa Cruz Biotechnology, Inc), BrdU (MCA2483, Bio-Rad, Hercules, CA, USA), NeuN (ABN78, Millipore Corporation), and Iba1 (019–19741, Wako Chemicals, Richmond, VA, USA). Sections were washed with PBST and incubated with fluorescent secondary antibodies (A11001, A11007, or A11037, Invitrogen, Carlsbad, CA, USA; CA94010, Vector Laboratories, Inc, Burlingame, CA, USA) for 2 hr at room temperature. Sections were mounted onto slides using a mounting medium (H-1200, Vector Laboratories, Inc) and imaged using a fluorescence microscope (Carl Zeiss Imager M1, Carl Zeiss AG, Oberkochen, Germany). Immunofluorescence was analyzed using the IMT i-Solution Inc 10.1 (17TH-5989 Walter Gage Rd., Vancouver, BC, CA) image analysis software.

The brain slices were incubated with primary antibody(goat anti-BDNF 1:150 abcam ab75040) at 4 °C overnight. For secondary antibody, the goat and rabbit two-step IHC reagent kits (Goldenbridge Beijing China) were used. The DAB were mixed before usage at 1:20 and incubated for 1–2 min.

Amyloid-β1–42 (Aβ1–42) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Before injection, the Aβ1–42 peptide was dissolved in a physiological saline solution at a concentration of 5 mg/ml and incubated at 37°C for 72 h to induce aggregation. Human full length BDNF (ab9794), NGF (ab138794) and NT-3 (ab138798) proteins and their corresponding rabbit polyclonal antibodies (ab75040, ab6198 and ab65804) were purchased from Abcam (Cambridge, UK). Rabbit monoclonal antibodies against phospho-ERK (#4370), total ERK (#4695), phospho-JNK (#4668), total JNK (#9258), phospho-p38 (#4511), total p38 (#9212) and GAPDH (#5174) were from Cell Signaling Technology (Beverly, MA, USA). Ceramide C6, an ERK activator, was obtained from Santa Cruz Technology (Santa Cruz, CA, USA). The ERK inhibitor PD98059 was from Sigma-Aldrich (St. Louis, MO, USA).