Rlsbad, CA, USA), 20 g/mL PEB (Merck Millipore, Darmstadt, Germany), 50 mg/L L-ascorbic acid (AA, Sigma-Aldrich Corp., St. Louis, MO, USA), 0.5 M FKL (Sigma-Aldrich Corp., St. Louis, MO, USA), 2 mM L-glut (Invitrogen Corp., Carlsbad, CA, USA), 5 HS (Invitrogen Corp., Carlsbad, CA, USA), 1?N2-supplement (N2; Invitrogen Corp., Carlsbad, CA, USA), 4 g/L D-(+)-glucose 10 (SigmaAldrich Corp., St. Louis, MO, USA), and 50 ng/mL NGF (Sigma-Aldrich Corp., St. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28151467 Louis, MO, USA). The culture myelination medium was renewed every 3 to 4 days. order Mangafodipir (trisodium) Cultures were kept for 28 days in vitro and treated as indicated from the sixth day after explantation until fixation, followed by staining.Immunocytochemistryand for MHCII, an additional 0.25 BSA. Cells were incubated for 1 hour at 37 (overnight at 4 for NF-L). After three washing cycles with PBS, the secondary antibody was applied for 1 hour at RT. The following secondary antibodies were used: Alexa FluorTM 594 goat anti-rabbit, Alexa FluorTM 594 mouse anti-rabbit, Alexa FluorTM 594 goat anti-rabbit (Invitrogen Corp., Carlsbad, CA, USA), 1:200 diluted in PBS and 1 BSA (SigmaAldrich Corp., St. Louis, MO, USA) and for NF-L 1:400 diluted in antibody diluent, followed by three washing cycles with PBS. Samples were embedded in 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) containing mounting medium (VectashieldTM, Vector Laboratories Inc., Burlingame, CA, USA) and analyzed with an upright fluorescence microscope (Nikon Eclipse TE200, Nikon AG, Tokyo, Japan and Axioplan 2 Imaging, Zeiss, Oberkochen, Germany).Real-time polymerase chain reactionFor immunocytochemistry, cells grown on glass cover slips were initially washed with phosphate-buffered saline solution (PBS) and fixed with 4 paraformaldehyde (PFA; Merck, Darmstadt, Germany) for 30 min for NF-L (neurofilament L) and 10 min for IL-17 receptor (IL-17R), following another washing step with PBS containing 1 bovine serum albumin (BSA; Sigma-Aldrich Corp., St. Louis, MO, USA). Samples were blocked using PBS-based blocking solution containing 10 (NF-L) or 4 (IL-17R) natural goat serum (NGS, DAKO, Hamburg, Germany) and 0.1 (NFL) or 0.2 (IL-17R) Triton X-100 (Merck, Darmstadt, Germany) for 30 min at RT. We used primary antibodies against IL-17 receptor A (IL-17R A; Abcam, Cambridge, UK), IL-17 receptor B (IL-17R B; Abcam, Cambridge, UK), and rabbit anti-NF-L (Millipore, Billerica, MA, USA), each diluted 1:400. Furthermore, antibodies against MHCI (1:750, mouse monoclonal antibody; Novus Biologicals, Littleton, CO, USA), MHCII (1:50, mouse monoclonal antibody; AbD Serotec Kidlington, UK) and transporter associated with antigen presentation (TAP) II (1:200, rabbit polyclonal; Bioss, Woburn, MA, USA) were used. Primary antibodies were diluted in PBS, containing 0.1 Triton (0.05 for the MHCI antibody), 10 NGS,Total cellular RNA was extracted using an RNeasyTM Mini Kit (Qiagen, Hilden, Germany) and quantified by NanoDrop-1000 (PEQLAB, Erlangen, Germany). Cells were washed twice with PBS and detached with buffer RLT. Total RNA (400 ng) was applied as matrix for cDNA synthesis using TaqManTM Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA) and High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer’s protocol (10 min at 25 , 120 min at 37 , and 5 min at 85 ). For subsequent real-time polymerase chain reaction (rtPCR) the thermal cycler (AbiPrism7000, Foster City,.
