Anti beta 3 tubulin

TAGs and normal glial cells acutely isolated by FACS were fixed in 4% PFA (Sigma-Aldrich) for 10 min, pelleted and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) for 4 min. Samples were pelleted again and blocked in 0.5% BSA (Sigma-Aldrich) for 15 min before immunostaining. Antibodies were diluted to 1:100; anti-GFAP (Dako), anti-Nestin (Abcam), anti-beta-tubulin III (Abcam), anti-MBP (Millipore) and 1:10; anti-O4 (RnD Systems) in 100 μl blocking buffer, and samples were incubated in 100 μl staining reaction for one hour at room temperature. Samples were washed in 3 ml blocking buffer, stained with Alexa Fluor 647-conjugated secondary anti-mouse and anti-rabbit antibodies (both from LifeTechnologies) in 100 μl for 45 min at room temperature and washed in 3 ml blocking buffer. Samples were resuspended in 100 μl 1xPBS before analysis, and cells stained with secondary antibodies alone or IgM isotype control (for O4, BD Biosciences) were used to set the gates. For cell cycle analysis, acutely isolated TAGs and glial cells from normal mice brains were fixed in 100% ice cold ethanol for 20 min, washed in 1xPBS and incubated for 30 min in RNase (1 mg/ml, Sigma-Aldrich) and propidium iodide (50 mg/ml, Sigma-Aldrich). All staining was acquired on AccuriC6 (BD Biosciences) and FlowJo (FlowJo, LCC, Oregon, USA) was used for analysis.

Cells were treated with either DMSO or ATRA, and then fixed with 4% paraformaldehyde. After permeabilization with TritonX-100 (0.1%), cells were incubated with primary antibody (anti-beta III Tubulin, Abcam) overnight at 4°C, washed with PBST (phosphate-buffered saline, 0.1% tween 20), and then incubated with Alexa Fluor 488 conjugated secondary antibody (Abcam) for 1 h at room temperature. DAPI (4′,6-diamidino-2-phenylindole) (Sigma Aldrich) was used for counter-staining. Fluorescent images were taken with Olympus IX53 inverted microscope.