In Situ Hybridization of RA Induced P19 Cells

P19 EC cells can be induced to differentiate into neurons in vitro when they were treated with 1x10(-6) mol/L retinoic acid. In the process of neuronal differentiation, P19 cells gradually migrate and form aggregates. Aggregated cells weakly adhered to the surface of cell culture dishes are usually dropped of during the procedure of in situ hybridization. To keep cells on the coverslips, we modified the procedure by using 1 g/L gelatin solution to cover the cells and substituting proteinase K treatment with pepsin treatment which digested cellular proteins mildly. With the treatment of pepsin, RNA probe can easily go through cell membrane and bind to its target, without loss of cell from coverslips.