These cells were differentiated by treatment with 10 M retinoic acid (Sigma, St. Louis, MO) added to the culture medium for 2 ?3 days or until they reached the desired confluency.Light microscopyCells were grown on coverslips to approximately 80 confluency. Cell media was decanted and the cells washed in 0.1 M sodium cacodylate buffer. The cells were then fixed in buffered 1.6 formaldehyde and 2.5 glutaraldehyde for 2? minutes at room temperature. The fixed cells were rinsed in 0.1 M sodium cacodylate buffer and stained with methylene blue in sodium borate solution for 1? minutes. The stained cells were rinsed with double distilled Millipore water. The cover slipsAndres et al. BMC Neuroscience 2013, 14:49 http://www.biomedcentral.com/1471-2202/14/Page 3 ofwere then inverted on a 1x3 positively charged slide. The cells were viewed and photographed at a magnification of 400X with an Olympus BX61 Microscope with NIKON photo assembly Digital Site DS-L1.Immunofluorescent stainingM17 cells were seeded onto 18 mm coverslips coated with poly-D-lysine (Sigma Aldrich). At described times, cells were fixed for 15 min in 3.7 formaldehyde, permeabilized with 0.1 saponin in PBS and blocked with 3 BSA (PBSS). Primary antibodies against the neuronspecific proteins 3-tubulin and synapsin-1/2 (Synaptic Systems, Gottingen, Germany) were diluted 1:1000 in PBSS and applied for 1 h at room temperature, followed by Alexa-conjugated secondary antibodies (Invitrogen) diluted 1:500 in PBSS. Coverslips were mounted onto slides with Prolong Gold DAPI (Invitrogen) and imaged using a Zeiss LSM 700 confocal microscope. Z-stack images were converted to a maximum projection image using Zen 2009 (Zeiss) software.SDS-PAGE and western blotting to assess levels of neuronal proteinsThe level of neuronal proteins and SNAP-25 in M17 cells was assessed by SDS-PAGE and Western blotting following the method described by Ray et al. [21]. Briefly, cells harvested in ice-cold physiological saline were lysed by incubating with a lysis buffer containing 10 mM Tris Cl, 150 mM NaCl, 1 mM EDTA, 1 mg/mL BSA, 1 mM PMSF, 1 Triton X-100 and protease inhibitors present in a protease inhibitor cocktail (cat. # P8340, Sigma, St. Louis, MO) which was included throughout the wash and solubilization steps to prevent protein degradation during the assay. The lysates were cleared by centrifugation at 16,000 x g for 10 min and immediately analyzed by SDSPAGE. Each gel (8 ?16.5 acrylamide) lane was loaded with approximately 30 g protein, which was determined by bicinchoninic acid (BCA) protein assay. Dry transfer to polyvinylidene fluoride (PVDF) membranes was performed using the iBlotTM System (Invitrogen, Capivasertib Carlsbad, CA) and blocked in 2 (w/v) bovine serum albumin for 1 hr (this and all subsequent incubations were performed in physiological saline, pH 7.5, room temperature). To determine the protein levels of specific neuronal proteins, blots were probed with primary antibodies overnight at 4 . The primary antibodies include neuron specific enolase (NSE; N6049), SNAP-25 (59682), and vimentin (V2258), all purchased from Sigma (St. Louis, MO); M1 muscarinic acetylcholine receptor (M1 mAChR, AB5164), choline acetyltransferase (ChAT, AB144P) from Cell Signaling (Boston, MA); synapsin (AB1543), neurofilament medium (145kDa, AB1981), and neurofilament heavy (200 kDa, AB1989) from Millipore (Billerica, MA); PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/7500280 and nicotinic acetylcholine receptor(nAChR-7, ab23832, Abcam, Cambridge, MA). Antibodies to.