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Observations of the fine structures of subcellular organelles and their pathophysiological changes have been made by many investigators by utilizing the many advantageous functions of confocal laser scanning microscopy (CLSM). However, reports of CLSM observations of fine inner structural changes of nuclei are rather rare. The usefulness and value of counterstaining in histochemical staining is widely appreciated. While observing methyl green (MG) nuclear-counterstained sections by CLSM, we found that fluorescence emitted through MG was clearly detected by CLSM. It is generally accepted that IVIG reacts specifically to double stranded (dbs)-DNA, as was proved by Umemura [30] in our laboratory. In the present study, MG-stained nuclei of the absorptive cells (terminal differentiating cells) of rat intestinal villi were observed by CLSM, and changes in the intensity and distribution of MG staining were examined. The small intestinal epithelial cells were also found to be slowly undergoing apoptosis. The apoptosis is thought to occur during the process of terminal differentiation, which takes about 48 hr. To examine those apoptotic changes, a TUNEL method, in which the 3'ends of fragmented dbs-DNAs are labeled with histochemically detectable dUTP, was employed. The interrelationship between cell functions, which can be detected by various histochemical observations of cytoplasmic substances, and cellular differentiation state, can be elucidated by using MG in CLSM.

Simultaneous Detection of DAB and Methyl Green Signals on Apoptotic Nuclei by Confocal Laser Scanning Microscopy