Quantitative analysis of microglial contamination in primary astrocyte cultures. (A) Triple immunostaining for primary astrocyte cultures using Iba1 (red), CD68 (green), and GFAP (blue) antibodies. Scale bar, 100 μm. (B) High magnification of the remaining Iba1-positive microglia after the shake-off method. Scale bar, 20 μm. (C) Triple immunostaining 4 and 14 days after the establishment of astrocyte primary cultures. Scale bar, 200 μm. (D) Quantification of the number of contaminating microglia in passaged culture. (E) Triple immunostaining of passaged culture using GFAP (green), BrdU (red), and Hoechst (blue) antibodies at 1, 4, and 14 days after seeding. Scale bar, 50 μm. (F) Changes in the number of BrdU-positive proliferating astrocytes to the total number of astrocytes over time in passaged culture. (G) Triple immunostaining of passaged culture using Iba1 (green) and BrdU (red) antibodies at 1, 4, and 14 days after seeding. Scale bar, 50 μm. (H) Changes in the number of BrdU-positive proliferating microglia to total microglia over time in passaged culture. (I-J) Triple immunostaining of passaged culture using GFAP (blue), CD68 (green), and Ki67 (red) antibodies at 1 and 10 days (I) and 14 days (J) after seeding. Scale bar, 50 or 200 μm. (K) The number of GFAP-positive astrocytes and CD11b-positive microglia in the Ki67-positive proliferating cell population. (L) Immunostaining of primary astrocyte cultures at 8 weeks after preparation showing Ki67-positive microglia. Scale bar, 200 μm. Inset shows high magnification image of Ki67-positive microglia. (M) Flow cytometric analysis at one and eight weeks after culture preparation showed increased number of CD11b-positive microglia. (N) Comparison of the number of the CD11b-positive population between one and eight weeks after preparation. *P <0.05, paired Student’s t-test. Data are presented as the mean ± SEM.