Isolation of Nucleoli

The human nucleolus is a prominent nuclear substructure assembled around tenderly repeated ribosomal genes (rDNA genes) on chromosomes 13, 14, 15, 21, and 22 and is the site of rDNA transcription and ribosome subunit synthesis. Visualized under electron microscopy, the nucleolus is separated morphologically into three distinct substructures: fibrillar centers (FC), which are surrounded by dense fibrillar components (DFCs), granular components (GC), and harvest cells by trpysinization. Rinse each dish three times with prewarmed PBS and, on removal of the last rinse, add 2 ml of trypsin-EDTA solution per dish. Swirl the dishes to make sure the trypsin-EDTA is distributed evenly and return the dishes to the incubator for about 5 mins. Resuspend the nucleoli with 0.5 ml of the S2 solution, followed by centrifugation at 1430 g for 5 min. To immunolabel purified nucleoli, spot about 5 ?l of the nucleolar suspension onto a polylysine-coated slide and air dry the spot. Rehydrate the slide in PBS for 5 min before carrying out a standard immunostaining procedure.