Sc 25269

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.

Following the collection of tissue, the samples were immersed in 4% paraformaldehyde for 48 h. Paraffin wax was used to embed them, which was then cut into sections of 5 μm. These sections underwent a gradient dewaxing and antigen retrieval using a citric acid solution. The activity of endogenous peroxidase was inhibited by applying 3% hydrogen peroxide for 15 min. Following rinsing, the sections were obstructed using a 3% BSA solution for 30 min. Subsequently, they were incubated with the primary antibody at 4 °C overnight. The next day, paraffin sections were brought to room temperature for 30 min and rinsed with PBS three times, each lasting 5 min. Afterwards, the second antibody was added gradually and left to incubate at ambient temperature for 50 min. After washing, DAB was added dropwise, and the reaction was terminated by microscopic control of color development time and rinsing after the appearance of positivity. Hematoxylin staining was rinsed after 1 min, and the hematoxylin differentiation solution was applied for several seconds. After dehydration and sealing, microscopic observation was performed, and data were collected. The following antibodies were used: PGP9.5(1:200, Cat# ab108986, Abcam), TH (1:200, Cat# sc25269, Santa Cruz), CHAT (1:200, Cat# ab137869, Abcam).

MN9D cells were seeded on a six-well chamber slide for treatment. The cells were then rinsed with PBS and fixed with 4% paraformaldehyde for 15 min at 26–28 °C. The cells were rinsed with PBS, then blocked and permeabilized with 1% normal goat serum and 0.5% TritonX-100 in PBS. The cells were then incubated with the following primary antibodies: mouse monoclonal anti-DYT5b antibody (1:200 dilution in blocking buffer, sc-25269, Santa Cruz Biotechnology), or rabbit polyclonal anti-DAT antibody (1:200 dilution in blocking buffer, sc-14002, Santa Cruz Biotechnology) overnight at 4 °C. The following day after three washes with PBS, the cells were incubated with appropriate secondary antibodies: DyLight 488 anti-rabbit IgG and DyLight 594 anti-mouse IgG (1:200, DI-1088, DI-2594, both Vector Labs, Burlingame, CA, USA) for 40 min at 26–28 °C. The cell nucleus was labeled with 4′,6-diamidino-2-phenylindole (DAPI) (C0065, Solarbio, Beijing, China) for 5 min. After three washes with PBS, the cover slips were sealed with Antifade Mounting Medium (P0128, Beyotime Biotechnology). The results were arranged and analyzed with Image-Pro Plus 6.