ABSTRACT
Protocol 8.1: Methyl green nuclear staining 214
Protocol 8.2: Haematoxylin and eosin staining (H&E) 215
Protocol 8.3: Feulgen staining 216
8.3 Determination of apoptosis by fluorescence microscopy
Protocol 8.4: DAPI staining 218
Protocol 8.5: AO staining 220
Protocol 8.6: TUNEL labelling 221
Protocol 8.7: Mitochondrial staining by CM-H2TMRos 222
8.4 Determination of apoptosis by electron microscopy 223
Protocol 8.8: Preparation of cells growing in Petri dishes for transmission electron microscopy
8.5 Location of cytochrome c during apoptosis 226
Protocol 8.9: Immunofluorescence detection of cytochrome c in cultured cells
Protocol 8.10: Pre-embedding immunogold labelling of intracellular antigens (cytochrome c) using ultrasmall gold probes
Protocol 8.11: Silver enhancement of gold labelling 231
Protocol 8.12: Localisation of cytochrome c in respiring mitochondria at the LM and EM level
8.6 Detection and localisation of lysosomal protease activity during apoptosis
8.7 References 235
8.1 Introduction
8.1.1 Defining modalities of cell death: the dichotomic view
is no longer valid
The concept of ‘programmed cell death’ was initially introduced by Lockshin to describe cell death that occurred in defined developmental niches and at predictable times during development, emphasising that cells are programmed to die during the normal development of the organism [1]. The first evidence that a genetic program existed for physiological cell death came much later from studying development in the nematode Caenorhabditis elegans [2-4] and the observation that the genes involved had mammalian counterparts [5-7].
