Cell Population Analyses of Neurospheres Generated In Vitro from Ovarian Cortical Cells in Mammals: Characterization by Immunohistochemistry and Flow Cytometry.

Abstract

Alternative experimental models based upon isolation and culture of extra-neural NSC/NPC or adult stem cells that can be induced in vitro to generate these cells and NS rely on development of procedures that can demonstrate the identity of cells as NSC/NPC and their compromise with the neural lineage during culture. Gene expression analyses by RT-PCRQ, immunohistochemical localization of characteristic NSC/NPC antigens, analyses of cell populations by flow cytometry, or ultrastructural analyses of in vitro generated NS are the most frequent and reliable methods to demonstrate the molecular and structural hallmarks of NSC/NPCs and NS. Among all these techniques, those aimed to immunolocalize specific NSC/NPC antigens on a cell basis and in a time-dependent fashion throughout culture require skillful NS processing before setting up the technique. In this chapter, comprehensive protocols for grouped OCC-NS preaggregation and paraffin embedding for immunolocalization of NSC/NPC antigens and NS population analyses by flow cytometry are fully explained.