Cyanine 5 (Cy5)-labeled dendrimer (D-Cy5) were administered intraperitoneally (20 mg/kg) to newborn mice at 6, 24 or 72 h after the hypoxic-ischemic brain injury. Animals were euthanized at 24 h after D-Cy5 administration and perfused with 10% formalin. Coronal brain sections (30 μm, 1:6 series) were cut using a Leica cryostat.
To evaluate the co-localization of D-Cy5 and astrocytes or microglia, brain sections were incubated overnight at 4 °C with chicken anti-glial fibrillary acidic protein (GFAP) (1:250, Abcam, MA. U.S.A.) and goat anti- calcium binding adaptor molecule 1 (IBA1) (1:250, Abcam, MA. U. S.A.). Sections were subsequently washed and incubated with fluorescent secondary antibodies (1:250; Life Technologies, MA, U.S.A.) for 2 h at room temperature. Next, the sections were incubated with 4',6-dia-midino-2-phenylindole (DAPI) (1:1000, Invitrogen) for 15 min to stain the nuclei. After washing, the slides were dried and cover slipped with mounting medium (Dako, Carpinteria, CA, USA). Confocal images were acquired with Zeiss ZEN LSM 710 (Zeiss, CA, U.S.A.) and processed with ZEN software.
To evaluate the co-localization of D-Cy5 and astrocytes or microglia, brain sections were incubated overnight at 4 °C with chicken anti-glial fibrillary acidic protein (GFAP) (1:250, Abcam, MA. U.S.A.) and goat anti- calcium binding adaptor molecule 1 (IBA1) (1:250, Abcam, MA. U. S.A.). Sections were subsequently washed and incubated with fluorescent secondary antibodies (1:250; Life Technologies, MA, U.S.A.) for 2 h at room temperature. Next, the sections were incubated with 4',6-dia-midino-2-phenylindole (DAPI) (1:1000, Invitrogen) for 15 min to stain the nuclei. After washing, the slides were dried and cover slipped with mounting medium (Dako, Carpinteria, CA, USA). Confocal images were acquired with Zeiss ZEN LSM 710 (Zeiss, CA, U.S.A.) and processed with ZEN software.