CSPG Expression on NPCs in vitro. (A-C) Immunofluorescence double staining of Nestin (A, red) and CSPGs (B, green) on neurospheres showed that almost all NPCs expressed CSPG (C, merge). The NPCs from E16 rat embryonic forebrains were cultured for 3 days in the NPC growth medium supplemented with EGF and bFGF. (D-F) The negative controls consisted of omission of the primary antibody. (G-I) The differentiated NPCs were double immunostained using anti-CS56 antibody (green) and anti-GFAP, βIII-tubulin and MBP antibodies (red) respectively. The result showed that CSPGs were expressed on GFAP+ astrocytes (G), βIII-tubulin+ neurons (H) and MBP+ oligodendrocytes (I). (J-M) The expression of CSPGs was observed on the dissociated NPCs culture in the growth medium containing EGF and bFGF (J, K), but diminished after Chase ABC (50 mU/mL) treatment for 48 hours (L, M). Hoechst 33342 (blue) was used to stain the nuclei. Scale bars: A~F, 100 μm; G~M, 50 μm.