2.5. Immunocytochemistry

Primary striatal neurons (DIV5-DIV10) were washed once with 1× PBS and fixed with 4% paraformaldehyde (Sigma-Aldrich, #158127) in 1 × PBS (37 °C, pH 7.4) for 15 min at RT. Subsequently, they were washed twice with 1 × PBS for 5 min. Permeabilization and blocking was performed with 0.1% Triton X-100 (Sigma-Aldrich; #X100-1L) and 10% donkey serum (PAN-Biotech, Aidenbach, Germany, #P30-0101) in 1 × PBS for 1 h. For immunostaining, the following antibodies were applied in a blocking solution overnight at 4 °C: chicken anti-GFP (1:1000; Abcam, Berlin, Germny, #ab13970), goat-anti-mTrkB (1:1000; R&D Systems, Minneapolis, MN, USA, #AF1494), rabbit anti-DARPP-32 (1:1000; Cell Signaling Technologies, Danvers, MA, USA, #19A3), mouse anti-ß-III-tubulin (1:1000; R&D systems, Minneapolis, MN, USA, #MO15013), goat anti-ChAT (1:200–500; Millipore, Burlington, MA, USA, #AB114), rat anti-Lamp-1 (1:20; Developmental Studies Hybridoma Bank, Iowa City, IA, # 1D4B-S; RRID: AB_528127), and sheep anti-mouse SORCS-2 (1:500; R&D systems, Minneapolis, MN, USA, #AF4237). After washing three times with 1XPBS for 10 min, primary antibodies were detected with the following secondary antibodies (incubation: 60–90 min at RT): donkey anti-chicken Alexa Fluor 488 (1:800; Jackson Immunoresearch, West Grove, PA, USA, #703-545-155), donkey anti-mouse Alexa Fluor 647 (1:800, Jackson Immunoresearch, West Grove, PA, USA, # 715-605-151), donkey anti-rabbit Cy3 (1:800; Jackson Immunoresearch, West Grove, PA, USA, #711-165-152), donkey anti-rabbit DyLight550 (1:200–400; Thermo Fisher Scientific, Schwerte, Germany, # SA5-10039), donkey anti-goat Alexa Fluor 647 (1:1500; Jackson Immunoresearch, West Grove, PA, USA, #705-605-003), donkey anti-rat Alexa 405 (1:800, Jackson Immunoresearch, West Grove, PA, USA, 712-475-15), and donkey anti-sheep Alexa Fluor 647 (1:500; Jackson Immunoresearch, West Grove, PA, USA, #2340703). Nuclei were stained with 0.4 mg/mL 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were again washed three times for 10 min with 1× PBS and the cells were mounted on Superfrost_Plus glass slides (25 × 75 × 1.0 mm; Thermo Scientific, Schwerte, Germany, #J1800AMNZ) using MERCK-FluorSave reagent (Merck, Darmstadt, Germany, #345789-20ML). For cell surface staining, after stimulation, the media was discarded and cold media with the goat anti-mTrkB (1:1000; R&D Systems, Minneapolis, MN, USA, #AF1494) antibody was added to the wells. The cells were incubated for 30 min at 4 °C. Afterwards, the cells were washed twice with ice-cold PBS and blocked for 1 h at RT. Then, incubation with the following other primary antibodies was performed: chicken anti-GFP (1:1000; Abcam; #ab13970), rabbit anti-DARPP-32 (1:1000; Cell Signaling; #19A3), mouse anti-GFAP (1:200; Millipore, Burlington, MA, USA, #MAB3402), and rabbit anti-GFAP (1:1000; GeneTex, Irvine, CA, USA, GTX108711).