Colony fixation, permeabilization, and blocking were performed as described (Motohashi et al., 2014 (link)). Primary antibodies, diluted in 0.5% BSA PBS, were then added and allowed to react at room temperature. After having been washed with PBS, the cells were stained with the secondary antibodies in the same manner. Primary antibodies: anti-mouse neuronal class III β-tubulin (1:500; TuJ-1, Covance), anti-mouse glial fibrillary acidic protein (GFAP, 1:500; Z0334, DakoCytomation), anti-mouse α smooth muscle actin (1:500; 1A4, Sigma), anti-mouse peripherin (1:100; MAB1527, Chemicon), anti-nestin (1:500; Rat401, Chemicon), and anti-S100β (1:100, Sigma, SH-B1). Secondary antibodies: Texas Red-conjugated anti-mouse IgG (1:500; Molecular Probes) and Alexa Fluor 488-conjugated anti-rabbit IgG (1:500; Molecular Probes). Nuclei were stained with Hoechst 33258 (Sigma). Colonies were examined by using an Olympus IX-71 fluorescence microscope.
ALP staining was performed with an ALP staining kit (Muto Chemical Co.). Regarding Alizarin Red staining, cells were fixed in methanol at 4°C for 20 min, and Alizarin Red (Kanto Chemical) staining was then carried out for 5 min at room temperature. For Oil Red O staining, cells were fixed in 4% paraformaldehyde for 15 min and then stained with 60% Oil Red O solution (Wako) for 30 min at room temperature.
ALP staining was performed with an ALP staining kit (Muto Chemical Co.). Regarding Alizarin Red staining, cells were fixed in methanol at 4°C for 20 min, and Alizarin Red (Kanto Chemical) staining was then carried out for 5 min at room temperature. For Oil Red O staining, cells were fixed in 4% paraformaldehyde for 15 min and then stained with 60% Oil Red O solution (Wako) for 30 min at room temperature.