Liposomal clodronate did not affect the proliferation or activation of astrocytes. (A, B) Immunostaining of primary astrocyte cultures 12 h after exposure to liposomal clodronate using GFAP (green) and nestin (red; A) or vimentin (red; B) antibodies. Scale bar, 50 μm. (C) Gene expression levels of GFAP and nestin for astrocytes with/without liposome-clodronate exposure as determined by qPCR. Astrocytes isolated from each condition by flow-cytometry were used in these analyses. There were no significant difference in the gene expression of GFAP and nestin between the two (n = 5, Mann–Whitney U test). (D-F) Proliferation of astrocytes at six hours after the exposure was assessed by immunostaining using pHis-H3 (red; D) or Ki67 (red; F) antibodies Scar bar, 100 μm. Quantification of the proportion of p-His-H3-positive astrocytes (E) and Ki67-positive astrocytes (G) to total astrocytes after liposome-clodronate treatment (n = 5, paired Student’s t-test). Data are presented as mean ± SEM.