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. 2020 May 1;9(5):1122. doi: 10.3390/cells9051122

Figure 2.

Figure 2

Immunocytochemical analysis of 3D spheroids. (A) Spheroids were fixed and cryosectioned then immunostained at weekly intervals from D2 until D42 stage. The first line represents the overview of the cryosectioned spheroids, while the rest of the panel shows higher magnifications. Relevant markers of proliferation (KI67), neural stem cells (NESTIN), neuronal differentiation (TUBB3 and MAP2), an intermediate filament of dendrites and axons (NF200), synaptic vesicles of neurons (VAMP2), astrocyte (AQP4), and oligodendrocyte (MBP) specific proteins were stained. Protein name IDs are indicated with colors, representing the color of the fluorophore used (e.g., green as Alexa 488; red as Alexa 594) Nuclei were counterstained with DAPI (in blue). Scale bar: 100 µm (first line only) and 25 µm. (B) Quantitative analysis of the immunostainings on confocal images. The numbers of Ki-67, NESTIN, AQP4, TUBB3, NF200kD, MBP, VAMP2, and MAP2 immunoreactive pixels were measured in 5 neurospheres (middle sections, 5 randomly selected fields/slide) at every time points. Data was normalized with DAPI positive nuclei number. Data were expressed as percentage of marker/DAPI ratio ± SEM (* p < 0.05).