Primary antibodies against the neuronspecific Title Loaded From File proteins 3-tubulin and synapsin-1/2 (Synaptic Systems, Gottingen, Germany) had been diluted 1:1000 in PBSS and applied for 1 h at space temperature, followed by Alexa-conjugated secondary antibodies (Invitrogen) diluted 1:500 in PBSS.10 M retinoic acid (Sigma, St. The fixed cells had been rinsed in 0.1 M sodium cacodylate buffer and stained with methylene blue in sodium borate option for 1 minutes. The stained cells have been rinsed with double distilled Millipore water. The cover slipsAndres et al. BMC Neuroscience 2013, 14:49 biomedcentral.com/1471-2202/14/Page three ofwere then inverted on a 1x3 positively charged slide. The cells have been viewed and photographed at a magnification of 400X with an Olympus BX61 Microscope with NIKON photo assembly Digital Site DS-L1.Immunofluorescent stainingM17 cells have been seeded onto 18 mm coverslips coated with poly-D-lysine (Sigma Aldrich). At described instances, cells had been fixed for 15 min in 3.7 formaldehyde, permeabilized with 0.1 saponin in PBS and blocked with three BSA (PBSS). Primary antibodies against the neuronspecific proteins 3-tubulin and synapsin-1/2 (Synaptic Systems, Gottingen, Germany) had been diluted 1:1000 in PBSS and applied for 1 h at space 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 employing a Zeiss LSM 700 confocal microscope.ten M retinoic acid (Sigma, St. Louis, MO) added towards the culture medium for 2 three days or until they reached the desired confluency.Light microscopyCells have been grown on coverslips to roughly 80 confluency. Cell media was decanted as well as the cells washed in 0.1 M sodium cacodylate buffer. The cells have been then fixed in buffered 1.6 formaldehyde and 2.five glutaraldehyde for two minutes at space temperature. The fixed cells had been rinsed in 0.1 M sodium cacodylate buffer and stained with methylene blue in sodium borate answer for 1 minutes. The stained cells were rinsed with double distilled Millipore water. The cover slipsAndres et al. BMC Neuroscience 2013, 14:49 biomedcentral.com/1471-2202/14/Page three 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 had been seeded onto 18 mm coverslips coated with poly-D-lysine (Sigma Aldrich). At described times, cells had been fixed for 15 min in 3.7 formaldehyde, permeabilized with 0.1 saponin in PBS and blocked with 3 BSA (PBSS). Key antibodies against the neuronspecific proteins 3-tubulin and synapsin-1/2 (Synaptic Systems, Gottingen, Germany) had been diluted 1:1000 in PBSS and applied for 1 h at space temperature, followed by Alexa-conjugated secondary antibodies (Invitrogen) diluted 1:500 in PBSS. Coverslips had been mounted onto slides with Prolong Gold DAPI (Invitrogen) and imaged working with a Zeiss LSM 700 confocal microscope. Z-stack photos were converted to a maximum projection image employing Zen 2009 (Zeiss) software.SDS-PAGE and western blotting to assess levels of neuronal proteinsThe amount of neuronal proteins and SNAP-25 in M17 cells was assessed by SDS-PAGE and Western blotting following the process described by Ray et al.