Immunofluorescence analysis of Musashi-1 was performed using 70% confluent log phase Neural Stem cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with Musashi-1 Monoclonal Antibody (14H1), eBioscience™ (Product # 14-9896-82) at 1 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Product # A-11006), (1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear and cytoplasmic localization. Panel e represents Neural Stem Cells differentiated to Astrocytes having no expression of Musashi-1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.