ABSTRACT
Protocol 3.1: FISH of interphase cells 47
3.4 Immunofluorescence microscopy and its applications
Protocol 3.2a: Fixation of cells and tissues for immunofluorescence microscopy
Protocol 3.2b: Antibody labelling of cells and tissues 51
Protocol 3.2c: Mowiol mounting medium 52
3.5 Electron microscopy techniques 54
Protocol 3.3: Resin embedding 55
Protocol 3.4a: Cryo-immunoelectron microscopy sample preparation
Protocol 3.4b: Immunolabelling of cryosections 59
Protocol 3.4c: Double labelling with colloidal gold, using colloidal gold with a mouse monoclonal and rabbit polyclonal antibody
3.6 Manipulation of the cell cycle 61
Protocol 3.5: Mitotic shake-off 62
Protocol 3.6a: Mitotic block and synchronisation 63
Protocol 3.6b: Double thymidine block 64
3.7 Biochemical analysis of nuclear processes 64
Protocol 3.7: Nuclear isolation from cultured cells 66
Protocol 3.8: Preparation of mitotic extracts 66
Protocol 3.9: Preparation of a nuclear lysate for immunoprecipitation and ELISA
Protocol 3.10: Electrophoretic analysis of molecular changes 70
Protocol 3.11: In gel digestion of proteins for proteomic analysis
3.8 References 73
3.1 Introduction
The interphase nucleus is the site of transcription and replication, DNA repair and recombination (Figure 3.1). The spectacular changes which occur during mitosis are only part of the dynamic capabilities of the nucleus. As new tools are developed to explore with higher resolution the relationships between structure and function, it is becoming clear that, like the cytoplasm, the nucleus contains a host of structures and bodies, functionally defined domains, a predictable organisation, and a dynamic response to cell cycle and metabolic stimuli [1].
