Anti tyrosine hydroxylase antibody

Bioprinted constructs were analyzed at day 30 using flow cytometry for the following markers: β-tubulin III (βT-III) (TUJ1) (R&D systems, Minneapolis, MN, United States). O4 (oligodendrocytes progenitor marker) (R&D systems, Minneapolis, MN, United States), Anti-Tyrosine Hydroxylase (TH) antibody (AbCam, Eugene, OR, United States), and GFAP (glial fibrillary acidic protein) (AbCam, Eugene, OR, United States) (a mature marker for astrocytes). The bioprinted constructs were degraded and the resulting cell suspension was processed as previously reported in see section “Assessment of Cell Viability Post Printing.” Briefly, the cell suspension was washed three times with PBS by centrifuging at 300 g for 5 min. The cell suspension was then fixed and stained per the manufacturer’s instructions (R&D Systems, Minneapolis, MN, United States). Isotype controls consisted of mouse IgG2A PerCP-conjugated Isotype control (R&D systems, Minneapolis, MN, United States), normal mouse IgM PE-conjugated Control (R&D systems, Minneapolis, MN, United States) and mouse IgG2b, kappa monoclonal [7e10g10] – Isotype control (AbCam, Eugene, OR, United States). The analysis was performed using the Guava EasyCyte HT flow cytometer (Millipore, Burlington, MA, United States).

Icariin (ICA, purity 98%) was bought from Nanjing Zelang Medical Technology, Co., Ltd. (Nanjing, China). 6-OHDA (CAS 28094157) was from Sigma Chemical, Co. (St. Louis, MO, United States). LPS (Escherichia coli strain O111:B4) was purchased from Calbiochem (San Diego, CA, United States). Anti-Tyrosine Hydroxylase (TH) antibody, Anti-CR3 complement receptor (OX-42), Anti-Iba1 antibody, Goat Anti-Mouse IgG H&L (TRITC), and Goat Anti-Rabbit IgG H&L (TRITC) were bought from Abcam (Cambridge, MA, United States). NF-κB signaling pathway antibodies were the products of Cell Signaling Technology (Beverly, MA, United States). Biotinylated secondary antibodies and vectastain avidin–biotin complex (ABC) kits were purchased from Vector Laboratories (Burlingame, CA, United States). ELISA kits were purchased from R&D Systems (Minneapolis, MN, United States). Griess reagent was obtained from Beyotime Biotechnology (Shanghai, China).

Primary cortical, DAergic neurons, and microglia were isolated from Sprague–Dawley rats (1 day pups). Briefly, rat brains were separated into cortex and midbrain and homogenized in Minimum Essential Medium (MEM, Gibco, NY, USA). The cell fractions were cultured in dishes containing MEM supplemented with 10 % fetal bovine serum (Gibco, NY, USA), 100 units/ml penicillin, and 0.1 mg/ml streptomycin (Gibco, NY, USA) in a humidified 5 % CO₂ chamber at 37 °C. Primary rat microglia were isolated using the “shaking off” method [25 (link)]. The purity of the primary neuronal cells was verified using specific marker protein expression, i.e., anti-neuronal nuclei (NeuN) antibody (1:200, Abcam, USA) for cortical neuronal cells, anti-tyrosine hydroxylase (TH) antibody (1:200, Abcam, USA) for DAergic neuronal cells, anti-microtubule-associated protein 2 (MAP2) antibody (1:200, Novus Biologicals, USA) for neuronal cells, and anti-dopamine transporter (DAT) antibody (1:200, Novus Biologicals, USA) for DAergic neuronal cells. The purity of the primary rat microglia was verified by flow cytometry (BD Biosciences, San Jose, CA, USA) at the Three-Dimensional Immune System Imaging Core Facility of Ajou University after staining with a specific antibody (CD11b/c, integrin αM; clone OX-42, Santa Cruz, CA, USA) (Supplementary Fig. 3).