Mouse monoclonal anti βiii tubulin

Primary neurons were cultured as described previously26 (link). Briefly, cerebral cortices were harvested from SD rat embryos (E 17.5). Then, the tissue was dissociated by trituration in DMEM containing 0.25% trypsin and 0.02% EDTA (Hyclone) followed by incubation for 10 min at 37 °C with agitation. DMEM with 10% FBS was added to neutralise the trypsin, and the solution was vortexed and then centrifuged at 2,000 rpm for 5 min. A portion of the cells were grown in Neurobasal medium (Gibco, Invitrogen Corp., USA) supplemented with 2% B27, 1% glutamine and 1% penicillin/streptomycin (Sigma-Aldrich Corp., USA) at 37 °C in a humidified incubator with 95% air and 5% CO₂. At 5 days post culture, the purity of the neurons and astrocyte was determined by immunocytochemistry using mouse monoclonal anti-βIII-tubulin (1:250; Millipore, Temecula, CA, USA) and rabbit monoclonal anti-GFAP (1:500; Abcam, USA) antibodies. At 7 days post culture, the purity of the neurons indicated that 95% of the cells in cultures were positive for βIII-tubulin (data not shown).

Primary antibodies used were: 1:200 rabbit monoclonal anti-CD34 (abcam, Cambridge, UK), 1:200 mouse monoclonal anti-CD34 (Thermo Fisher Scientific, Waltham, MA, USA), 1:300 mouse monoclonal anti-Iba1 (Millipore, Burlington, MA, USA), 1:200 mouse monoclonal CD68 (abcam, Cambridge, UK), 1:250 rat polyclonal anti-CD11b (BD Biosciences, Franklin Lakes, NJ, USA), 1:200 rabbit polyclonal anti-Ki67 (abcam, Cambridge, UK), 1:400 mouse monoclonal anti-GFAP (Sigma, St. Louis, MO, USA), 1:300 mouse monoclonal anti-S100β (Sigma, St. Louis, MO, USA), 1:300 mouse monoclonal anti-misfoldedSOD1 (MediMabs, Montreal, Northern Province, Canada), 1:400 mouse monoclonal anti-βIII-Tubulin (Millipore, Burlington, MA, USA). Secondary antibodies used were: 1:500 goat anti-rabbit-AlexaFluor546 or AlexaFluor633 (Thermo Fisher Scientific, Waltham, MA, USA), 1:500 goat anti-mouse-AlexaFluor488, AlexaFluor546, or AlexaFluor633 (Thermo Fisher Scientific, Waltham, MA, USA), 1:500 AlexaFluor633 (Thermo Fisher Scientific, Waltham, MA, USA).