Localization of Apoptotic Cells in the Human Epidermis by an In Situ DNA Nick End-labeling Method Using Confocal Reflectant Laser Microscopy

Abstract

We describe an immunohistochemical method that allows the detection of apoptotic cells in human epidermis by use of confocal laser reflectance and antibody-immunogold-silver complexes. For this purpose, the site of free 3′-OH DNA ends was detected by the reflectance from heavy metal products (anti-digoxigenin antibody-immunogold-silver complexes) instead of 3, 3′ -diaminobenzidine (DAB) reaction products in the conventional in situ nick end-labeling of DNA strand breaks (ISEL) technique. Localization of double-stranded DNA was demonstrated by the autofluorescence of methyl green. The ISEL technique using confocal reflectant laser microscopy (CRLM) clearly showed the most intense reflectance in the nuclei of granular cells, in contrast to only a weaker reflectance in those of basal cells. On the other hand, the extent of autofluorescence of methyl green was significantly more intense in the nuclei of basal cells and showed a reciprocal relation to that of the reflectance. Therefore, granular cells were most prone to apoptosis and did not contain double-stranded DNA, as indicated by the lack of stainability with methyl green. In addition, this method demonstrating the simultaneous localization of both free 3′-OH DNA ends and double-stranded DNA proved to have a wide range of applications, including the study of other DNA autolytic processes.