Following 90d, animals were euthanized, perfused transcardially and their brains removed and post-fixed. Serial 30μm sections (every 10th section) were stained (0.1% cresyl violet acetate). Prussian blue staining for SPIO containing cells was performed (17 (link)).
Free-floating sections were blocked with 10% normal goat serum (NGS; Invitrogen, Carlsbad, CA) for 10 min. Primary antibodies were diluted in 0.1M PBS containing 10% NGS and 0.1% triton x-100 and used at the following concentrations: rabbit anti-human glial fibrillary acidic protein 1:200 (GFAP; Abcam, Cambridge, MA); mouse anti-human nestin 1:100 (Abcam); mouse anti-human cyclic nucleotide phosphodiesterase 1:100 (CNPase; Millipore, Temecula, CA); mouse anti-human nuclei 1:100 (Acris Antibodies, Germany); and mouse anti-rat GFAP 1:300 (Sigma-Aldrich, St Louis, MO). Secondary antibodies (1:1000; Invitrogen) included: goat anti-rabbit AlexaFluor (488 or 568 nm) or goat anti-mouse AlexaFluor (488 or 555 nm). Sections were air-dried and coverslipped with VectaShield anti-fade mounting media (Vector Labs, Burlingame, CA). Slides were stored at 4°C and immunolabeled slides were scanned on a confocal microscope (BioRad 1024). Z-series of 10–12 images were collected by stepping through ~1μm sections for each tissue.
Free-floating sections were blocked with 10% normal goat serum (NGS; Invitrogen, Carlsbad, CA) for 10 min. Primary antibodies were diluted in 0.1M PBS containing 10% NGS and 0.1% triton x-100 and used at the following concentrations: rabbit anti-human glial fibrillary acidic protein 1:200 (GFAP; Abcam, Cambridge, MA); mouse anti-human nestin 1:100 (Abcam); mouse anti-human cyclic nucleotide phosphodiesterase 1:100 (CNPase; Millipore, Temecula, CA); mouse anti-human nuclei 1:100 (Acris Antibodies, Germany); and mouse anti-rat GFAP 1:300 (Sigma-Aldrich, St Louis, MO). Secondary antibodies (1:1000; Invitrogen) included: goat anti-rabbit AlexaFluor (488 or 568 nm) or goat anti-mouse AlexaFluor (488 or 555 nm). Sections were air-dried and coverslipped with VectaShield anti-fade mounting media (Vector Labs, Burlingame, CA). Slides were stored at 4°C and immunolabeled slides were scanned on a confocal microscope (BioRad 1024). Z-series of 10–12 images were collected by stepping through ~1μm sections for each tissue.
