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

Abstract

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 MG 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.

A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green

“…This fact, together with the lack of a generalized availability of spectral confocal microscopes, probably hindered Mg's notable properties as a fluorescent stain. We could find only one report in the literature on the use of Mg as a fluorescent Dna stain on histological preparations (Itoh et al 2003). In this case, however, a high concentration of Mg was used to generate the usual green stain visible in bright field microscopy, and the fluorescence excitation-emission wavelengths were coincident with those of orange-red fluorophores such as TrITC hence very different to the advantageous properties reported in the present work.…”

Histopathological Review of Silent Pituitary Somatotroph Adenoma

“…These paradoxical responses of GH in TRH or GnRH provocation test, which were used as a hallmark of silent somatotroph adenoma in our previous reports [10][11][12][13], are observed primarily in densely granulated somatotroph adenomas. Subcellular organelles and diaminobenzidine signals of immunohistochemical staining can be observed under confocal laser scanning microscopy (CLSM) [2][3][4]. Therefore, when tumor specimens were not available for electron microscopy, CLSM can detect secretory granules using paraffin sections immunostained with anti-GH antibody …”