Immunocytochemical Analysis of hBM-MSCs

A standard immunocytochemical protocol was used as previously described [40 (link), 45 (link), 47 (link)–49 (link)]. Histone H2B-GFP-transfected hBM-MSCs were plated onto collagen IV (Sigma‒Aldrich)-coated plastic or glass coverslips and maintained in neural induction media. Cells were rinsed with PBS and fixed in freshly prepared 4% paraformaldehyde (PFA; Sigma‒Aldrich). Fixed cells were blocked for 2 h in PBS containing 10% normal horse serum (Gibco) and 0.25% Triton X-100 (Sigma) and incubated overnight at 4 °C with antibodies against β-III-tubulin (TUJ1; 1:500, Covance), fibrillarin (1/300, Abcam) and lamin A/C (1/300, GeneTex) in PBS containing 1% normal horse serum and 0.25% Triton X-100. The next day, the cells were rinsed and incubated with secondary antibodies conjugated with Alexa Fluor® 488 (anti-rabbit; 1:500, Molecular Probes) and Alexa Fluor® 594 (anti-mouse; 1:500, Molecular Probes). Cell nuclei were counterstained with DAPI (0.2 mg/ml in PBS, Molecular Probes). Alexa Fluor 488® phalloidin (Molecular Probes) was used to selectively stain F-actin. Data are representative of ten independent experiments per condition.