The EGFP-hADSC-NCs were tracked and identified by immunohistochemical staining with the neuron markers MAP2 (Synaptic Systems, Cat#188011 and Cat#188003), NF-200 (Cell Signaling, Cat# 2836), NeuN (Sigma, Cat# SAB4300883), Synapsin1/2 (Cell Signaling, Cat#5297) Human Nuclear Antigen (HuNA, Merck Millipore, Cat# MAB1281), the proliferative marker Ki67 (R&D Systems, Cat#AF7649), astrocyte marker of GFAP (Synaptic Systems, Cat#173011), microglia marker of IbaI (FUJI FILM, Cat# 019-19741), the cell apoptosis marker Caspase 3 (Cell Signaling, Cat# 9579S) and blood vessel endothelial marker of CD31(R&D Systems, Cat#AF3628) by following previously published methods [72 (link)]. Briefly, after the Morris water maze test, mice from each group were sacrificed, and intracardial perfusion was performed. Then, the brains were removed, fixed with 4% paraformaldehyde and dehydrated with a sucrose gradient. Brain sections 20 μm in thickness were obtained with a cryostat (Leica, CM1850), followed by immunohistochemical staining. The distribution of the EGFP-labeled hADSC-NCs in specific brain sites was observed through confocal microscopy (Leica, SP8). Additionally, colocalization was carefully observed to determine whether any transdifferentiation of hADSC-NCs into neuronal cells had occurred.
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