Visualization of microtubules of cells in situ by indirect immunofluorescence

Abstract

Microtubule staining patterns can be visualized within cells in situ on the surface of fish scales from the squirrel fish, Holocentrus ascensionis, and the common goldfish, Carassius auratus, after incubation with antibodies to sea urchin tubulin and fluorescein-labeled goat antibodies to rabbit immunoglobulin G. Chromatophores in situ from both species reveal a radial microtubule framework that orients the alignment of pigment granules. Innervating fibers of erythrophores on the H. ascensionis scale can also be observed. In situ, pseudo-epithelial cells called scleroblasts are few and form a simple radial framework with a localized microtubule organizing center (MTOC). Microtubules in scleroblasts in vitro emanate from localized MTOCs but are much less radially organized than in situ. Scleroblasts in situ on the scale of C. auratus show microtubules that curve abruptly into coalignment with phase striations on the fibrillary plate. The phase striations arise from the orthogonal plies of collagen in intimate associations with the scleroblasts. The role of microtubules in scleroblasts may thus be to provide orientation for collagen fibrillogenesis, analogous to their role in the orientation of cellular fibers in plants. That cells in situ exhibit highly related and coordinated microtubule staining patterns reaffirms that the cytoskeleton plays an important role in the organization of differentiated tissues.