Ab150346

For brain slices, immunofluorescent staining shared a same procedure with immunohistochemistry staining before secondary antibodies incubation. For cellular cultures, sterile coverslips were placed in 24-well plates and coated with PLL before cells were plated on the coverslips. After intervention, cells were first washed with PBS and fixed with 4% PFA. Then cells were permeablized with 0.2% Triton X-100 and blocked with 5% BSA. Incubate brain slices or cell slides with following primary antibodies overnight at 4°C: anti-DOPA decarboxylase (AADC) antibody (1:250, Abcam, ab3905), anti-dopamine transporter (DAT) antibody (1:50, Santa Cruz, sc-32258), anti-TH antibody (1:750, Abcam, ab129991), anti-MAP2 antibody (1:500, Servicebio, Wuhan), anti-GABA antibody (1:200, Abcam, ab8891), anti-dynorphin antibody (1:50, Santa Cruz, sc-46313), anti-enkephalin (1:100, Abcam, ab150346). After being washed, sections were incubated in dark with an appropriately diluted Alexa 488- or Cyanine 3-coupled secondary antibodies followed by DAPI staining nucleus for 10 min. Coverslips were mounted on slides with one drop per coverslip of antifade mounting medium (Beyotime, China). Images were collected using fluorescence microscopy with image manipulation software, and analyzed using Image-Pro Plus software.

Protein was extracted from tissues via RIPA (Beyotime, Beijing, China). The protein concentration was assessed by BCA kit (Beyotime). Then, protein samples were separated through SDS-PAGE, which was transferred onto PVDF membrane. The membrane was blocked by 0.5% skimmed milk for 2 h at room temperature, followed by incubation with primary antibodies against Protein Penk (1:1000; ab150346; Abcam, United States), C3 (1:1000; ab181147; Abcam), Fga (1:2000; ab108616; Abcam), Slc4a1 (1:1000; ab196798; Abcam), and β-actin (1:200; ab115777; Abcam) at 4°C overnight, and secondary antibodies (1:5000; ab7090) at room temperature for 2 h. Protein blots were analyzed through the Western Lighting Ultra (Thermo).