Z0334

For brain tissue preparation, mice were deeply anesthetized with Zoletil (12.5 mg/kg) and Rompun mix (17.5 mg/kg) administered intraperitoneally. Mice were perfused transcardially with heparin dissolved in PBS (pH 7.2). The dissected brain tissues were frozen at -80 °C for Western blotting. Tissues were homogenized with total RNA and protein isolation kit (NucleoSpin® RNA/Protein #740933.50, Macherey-Nagel, Dűren, Germany). The protein samples were quantified with Pierce™ BCA Protein Assay Kit and 50 μg protein sample were used for each Western blot. The primary antibodies were applied in the following concentrations: anti-GFAP (rabbit, 1: 1,000; Dako #Z0334), anti-Iba1 (rabbit, 1:300; Wako #NB100-1028), anti-EAAT1 (rabbit, 1:1,000; Santacruz # sc-15316), anti-EAAT2 (rabbit, 1:1,000; Cellsignaling #3838), anti-Cx43 (mouse, 1:1,000; Santacruz #sc-59949), anti-PSD95 (mouse, 1:2,000; Thermo #MA1-046), anti-NeuN (mouse, 1:1,000; Millipore #MAB377) anti-GAPDH (rabbit, 1:10,000; Abfrontier #LF-PA0018). Secondary antibodies were conjugated with horse-radish peroxidase (HRP) (1: 10,000, Invitrogen). The HRP signals were visualized using an enhanced chemiluminescent (ECL, Abfrontier #LF-QC0101, Gyeonggi-do, Korea) substrate.

Brains from Tardbp^+/+ and Tardbp^+/Q101X male mice (both n = 3) were harvested for histopathological analysis at one year of age. Mice were perfused transcardially with 0.9% saline followed by 4% paraformaldehyde (PFA). Following dissection, brains were transferred to formalin and subsequently embedded in paraffin wax. Sections were stained with haematoxylin and eosin, and antibodies against GFAP (3 ug/mL, DAKO, Z0334), IBA-1 (1.7 ug/mL, WAKO Chemicals, 019-19741), p62 (5.6 ug/mL, Progen Biotechnik, GP62-C), MBP (2 ug/mL, Covance, SMI-94R) and neurofilament (0.5 ug/mL, Sigma, N5389) using a Ventana automated immunohistochemical staining machine (Ventana Medical Systems, Tuscon, USA). Once incubated with appropriate secondary antibodies, immunoreactivity was developed with 3,30-diaminobenzidine and counterstained with haematoxylin.

Tissues for immunofluorescence were fixed in 4% PFA solution for 24 h and then immersed in 30% sucrose for 24 h twice. Processed brain tissue was coronal sectioned at a thickness of 30 μm in four series (8–10 slices per series). Each series was incubated with primary antibodies against TH (1:500, Millipore AB152), DAT (1:100, Millipore MAB369), GFAP (1:300, DAKO Z0334), or Iba1 (1:500, Wako 019-19741), respectively, at 4 ℃ overnight, followed by secondary antibodies incubation at room temperature for 2 h. Images were obtained by confocal microscope (NiE-A1 plus, Nikon) or fluorescent microscope (E80i, Nikon) and processed by ImageJ (1.49c). The number of TH-positive cells was quantified within area of compact part of SN. The density of DAT signal was quantified by sampling 5 repeated 50 × 50 pixel areas within striatum. All quantification was single-blinded.