Figure 5

A. Double immunolabeling and CLSM analysis of NF1 protein with NF1GRP(D) antibody and cytoskeletal marker proteins at 13 weeks EGA in developing human epidermis. (a) NF1 protein (green) and cytokeratin 14 (red) show colocalization (yellow) on many locations of the basal cells. (d) High magnification reveals a marked degree of colocalization of NF1 protein and cytokeratin 14 labeling patterns. (b) NF1 protein (red) and actin (green) antibodies show separate labeling patterns. (e) Higher magnification reveals that the NF1 positive fibrils (red) are located closer to the nuclei (n), while actin (green) is located more peripherally. (c) NF1 protein (red) and α-tubulin (green) antibodies show separate labeling. (f) High magnification of labeling for α-tubulin and NF1 protein. Scale bars 10 μm in a-c, 1 μm in d-f. B. Double immunolabeling and CLSM analysis for NF1 protein with NF1GRP(D) antibody and desmoplakin or β4 integrin in developing human epidermis at 13 weeks EGA. (a) Desmoplakin (red) is present in all epidermal cell layers, although the basal cells show weaker labeling. Instead, NF1-specific antibody labels the basal and the periderm cells. (c) Higher magnification reveals that cytokeratin fibrils associated with NF1 protein (green) terminate to spots of desmoplakin (red) indicating the location of desmosomes. Insert in (c) show the location of a desmoplakin dot (red) at the end of a NF1 positive fibril (green). (b) β4 integrin (red) label the basal aspect of the basal cells, while NF1 protein (green) label the basal cell cytoplasm. (d) Higher magnification reveals that β4 integrin dots (red) are located at the end of NF1 positive fibrils (green). Scale bars 10 μm in a, b; 2 μm in c, d. C. Double immunolabeling and CLSM analysis of NF1 protein with adherens junction proteins and α-actinin in developing human epidermis at 13 weeks EGA. (a) E-cadherin antibody (green) labels the cell-cell junctions of the basal and intermediate cells. NF1GRP(D) antibody (red) show basal and periderm cell cytoplasmic labeling. (d) Higher magnification reveals that immunosignals for E-cadherin (green) and NF1 protein (red) are not colocalized. (b and e) Also α-actinin (green) and NF1 (red) are seen mutually exclusive. (c) β1 integrin (green) labels the cell membranes of the basal cells, while NF1 protein (red) labeling is cytoplasmic. (f) In higher magnification, NF1 positive fibrils (red; arrowheads) are terminating between the β1 integrin labeling (green) at cell-cell junction sites. n, nucleus. Scale bars 10 μm a-c; 1 μm in d-f.