Laser Microdissection Dissected cerebella were frozen using isopentane cooled by liquid nitrogen and stored at ?80 C. Cav3.1 (encoded by after VEGF treatment. 2. Materials and Methods All procedures were carried out under established requirements of the German federal state of North Rhine Westphalia, in accordance with the European Communities Council Directive 2010/63/EU on the protection of animals utilized for scientific purpose. 2.1. Enriched PC Culture The procedure to obtain a highly real PC culture was already explained before [32]. Briefly, anti-GD3 ganglioside antibodies were obtained with the help of a R24 hybridoma cell collection (hybridoma R24, HB-8445, ATCC, Manassas, VA, USA). On day two, eight petri dishes for the immunopanning process (351007, Corning Falcon, Corning, NY, USA) were prepared with 3 mL of 50 mM Tris-HCl (pH 9.5), 24 L of goat anti-mouse IgG antibody (31160, Thermo Fisher, Waltham, MA, USA), and 60 L goat anti-mouse IgM antibody (ab9167, Abcam, Cambridge, UK). On day three, prepared petri dishes were washed with filtered phosphate-buffered saline (PBS, pH 7.4), and the anti-GD3 supernatant as well as the anti-Thy1.1 antibody were added. Cerebella were obtained from male and female Wistar rat pups of p0. Digesting of the tissue was performed with a sterile filtered answer including: 5 mL Earles answer with EDTA and NaHCO3, 15 L of DNase (5 mg/mL), 1.2 mg of L-Cysteine (168149-2.5G, Sigma-Aldrich, St. Louis, MO, USA), 60 L of papain answer (31.43 mg/mL, PAPL, Worthington LS 003119 in 60 L of papain activation solution (6.66 mg L-Cystein, 44 TAPI-1 L 0.25 M EDTA solution, 47.16 L Mercaptoethanol in 10 mL of ddH2O)). Dissected cerebella were incubated in this answer for 1 h at 35.5 C. After incubation, cerebella were triturated and a centrifugation step with Percoll gradients was performed, as explained before [32]. Afterwards, the PCs were isolated with the help of anti-GD3 and anti-Thy1.1 antibodies. The cells were counted using a Neubauer chamber, and 100.000C120.000 cells were plated in 96er well plates in serum-free medium. Serum-free medium was replaced the day after extraction and then every second day. VEGF (V4512, Sigma-Aldrich, c = 0.1 g/mL in ddH2O) was mixed with serum-free medium and applied to the group of stimulated PCs. 2.2. Laser Microdissection Dissected cerebella were frozen using isopentane cooled by liquid nitrogen and stored at ?80 C. Removable parts of the cryostat were cleaned with a solution made up of 1 mM EDTA and 0.1 M NaOH in DEPC-treated water to inhibit RNases. After fixation with tissue freezing medium (No. 14020108926, Leica, Wetzlar, Germany), 12 m sections were obtained with the help of the cryostat (Leica microsystems CM3050 S, Wetzlar, Germany). Sections were mounted onto RNase-free polyethylene naphthalate-(PEN)-membrane slides manufactured for LMD (No. 11505151, Leica) and dried on a heater at 40 C for at least 20 min. Tissues were stained by applying a solution made up of 1% methylene blue, 1% azure II, and 1% Borax in DEPC-treated water and washing with DEPC afterwards, as explained by Pieczora et al. [33]. Slides were microdissected using the LMD6500 system (Leica Microsystems, TAPI-1 Wetzlar, Germany). Then, 1,000,000 m2 of enriched PCs, which equals 2000 PCs, and 1,000,000 m2 of the molecular layer from 20 female and TAPI-1 male Wistar rats were isolated in total and samples were collected in 20 L of lysis answer (AM1931, Invitrogen, Waltham, MA, USA) each. All samples were stored at ?80 C. 2.3. RT-qPCR For total RNA isolation from cultured cells, NucleoSpin? miRNA isolation Mouse monoclonal to KSHV ORF26 kit (No. 740304, Macherey-Nagel, Dren, Germany) was used according to the manufacturers protocol. Briefly, pelleted cells were resuspended in 300 L ML lysis buffer. Then, 100% EtOH was added, and samples were loaded onto the NucleoSpin RNA Column and centrifuged briefly. After washing with different buffers and incubation with rDNase, the RNA columns were washed with buffer MW 1 and MW 2 and then the total RNA was eluted in 30 L of nuclease-free water and stored at ?80 C. For the isolation of the total mRNA of the LCM samples, we used a RNAqueous-Micro Kit for LCM samples (AM1931, Invitrogen) according to the manufacturers protocol. Briefly, wash solutions were prepared, and the elution answer was heated to 95 C. Dissected PC samples were stored in lysis answer at ?80 C, and 100 L of lysis solution containing the cells TAPI-1 were incubated, and to recover large and small RNAs, 129 L of 100% ETOH were added. After three washing actions, the RNA was eluted by using 10 L of preheated elution answer. To inactivate DNases, the DNase protocol TAPI-1 of the RNAqueous-Micro Kit for LCM samples was performed after the RNA extraction process. In the end, the RNA was stored at ?80 C. cDNA was synthesised using qScript cDNA SuperMix (95048-100, Quantabio, Beverly, MA, USA). Therefore, 20 L samples consisting of 10 L undiluted total RNA, 4.