Class III β‐Tubulin isotype (β III) in the adrenal medulla: III. Differential expression of neuronal and glial antigens identifies two distinct populations of neuronal and glial‐like (sustentacular) cells in the PC12 rat pheochromocytoma cell line maintained in a gelfoam matrix system

Background: The rat PC12 pheochromocytoma cell line provides an established system for the study of neuronal differentiation. To our knowledge, glial differentiation has not been reported in this cell line. Methods: We have studied, by immunohistochemistry and immunoblot- ting, the presence of neuronal cytoskeletal antigens (class III b-tubulin isotype (b III), microtubule associated proteins MAP2, MAP1B and tau, and different neurofilament (NF) protein components), and synaptophy- sin in comparison with the glial fibrillary acidic protein (GFAP) and S-100 protein in the PC12 cell line. In three different experiments, PC12 cells were maintained in a three-dimensional gelatin foam (Gelfoam) matrix system for up to 34 days with and without treatment with 1 mM dibutyryl cyclic (dc)AMP. Immunohistochemistry was performed on explants rang- ing from 2 to 32 days-in vitro, which were fixed in either Bouin's solution, 70% ethanol, or 10% neutral-buffered formalin and embedded in paraffin. Immunoblotting was performed on Gelfoam explants with a panel of antibodies against all aforementioned neuronal and glial markers. Addi- tional immunoblot experiments using anti-GFAP and anti-b III monoclo- nal antibodies in cell suspensions and homogenates from PC12 monolayer cultures were carried out to compare growth conditions in relation to the expression of these proteins.