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. 2000 Nov 28;97(25):13883–13888. doi: 10.1073/pnas.250471697

Figure 4.

Figure 4

Alkaline phosphatase (AP) enzyme histochemistry and immunocytochemistry of cells and neurospheres derived from Gtv-a transgenic mouse astrocytes infected with RCAS-alkaline phosphatase, showing cells expressing both AP and neuronal phenotype markers. (a) P2 astrocyte monolayer after infection with the avian leukosisvirus expressing the AP reporter gene, showing infected astrocytes (e.g., arrow). Upper right Inset shows AP histochemistry of the DF1 chicken embryo fibroblast line engineered to produce the RCAS-AP leukosisvirus. Lower right Inset shows a single infected astrocyte with both AP histochemical labeling (blue-black punctae) and GFAP immunofluorescence (green, FITC). Scale bars = 25 μm in a, 30 μm in both Insets. (b) A neurosphere derived from a Gtv-a astrocyte monolayer. AP histochemistry reveals cells of this neurosphere expressing the RCAS-AP gene, thus indicating derivation of the clone from a single, infected astrocyte. Upper right Inset shows an example of a neuron derived from such a neurosphere, immunofluorescence for β-III tubulin (green, FITC). Lower pair of Insets show the same neurosphere expressing AP (left) and MAP-2 (right), revealing numerous MAP-2-positive processes emanating from an RCAS-infected neurosphere. Scale bars = 100 μm in b, 50 μm in the lower Insets, and 40 μm in the upper Inset. (c and d) A single RCAS-AP infected neuron that has migrated away from its neurosphere and differentiated. Brightfield labeling of AP reaction product (blue dots) in this neuron (c), colocalized with β-III tubulin immunofluorescence (FITC green) shown in d. Asterisk marks the nucleus of this cell in each figure. Scale bars in c and d = 10 μm. (e) Low (e) and high (lower Inset) magnification of an RCAS-AP-infected neurosphere showing β-III tubulin-positive neurons (FITC, green) within a densely AP-positive neurosphere. Arrow points to double labeled neurons within the neurosphere. Asterisks mark the top edge of the neurosphere. Scale bar = 10 μm. (f and g) A double labeled neuron at the edge of a neurosphere (the neurosphere is in the upper left portion of the field), as seen in single exposures of the same field, brightfield for AP in f and immunofluorescence for β-III tubulin in g. The asterisks are within the nucleus of this double labeled cell in both images, and other nuclei appear as sparsely positive AP ovals. Scale bars = 10 μm.