The histochemical distribution of protein bound sulfhydryl groups in human epidermis by the new staining method.

Abstract

Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) which is nonfluorescent by itself but will react readily with-SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of-SH groups and S-S linkages in the human epidermis. The distribution of-SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S-S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared

<b>DISTRIBUTION OF -SH AND S-S IN EPIDERMIS AND ECCRINE GLANDS: COMPARISON OF THE FINDINGS BASED ON THE ROUTINE AND <i>N</i>-(7-DIMETHYLAMINO-4- METHYL-3-COUMARINYL)MALEIMIDE (DACM) </b><b>STAININGS </b>

“…Epz'dermz's -SH groups were distributed in the epidermis as reported by Ogawa er a/. (9). The fluorescence of -SH groups by DACM staining was observed to be weak in the basal cells, moderate in the prickle cells and strong in the lower horny cells (Fig.…”

“…S-S linkages were present in the horny layer and not in the living layers of epidermis, as described previously (9,12). Comparison of the S-S distribution pattern (Fig.…”

“…DACM has a very high sensitivity to -SH groups and can visualize a very small amount of -SH groups with fluorescence (9). Therefore, it is likely that there are no differences in the distribution patterns of -SH groups and S-S linkages by DACM staining when comparing 49 frozen and deparaffinized sections, even though formalin fixes tissue proteins containing -SH groups and S-S linkages in an equal degree.…”

“…In the horny layer, the -SH fluorescence of the MIC parts appeared brilliantly in the lower layer and gradually decreased in degree upward through the horny layer. The fluorescence of the cytoplasm in the keratinized horny cells was faint (9,10). The localization of S-S linkages was not characteristic of the living layers, but appeared abruptly at the junction of the living and horny layers.…”

“…(9,10) developed a new hist.ochemical staining method for sulfhydryl groups using DACM, the characteristic distribution and mobility of-SH groups and S-S linkages have been studied in human epidermal layers during keratinization (9,10), a human epidermal cancer (8) and human hair, dermis and cutaneous appendages (13). In these studies, unfixed frozen sections were used and the distribution of -SH groups and S-S linkages has clearly appeared in those tissues under fluoromicroscopes.…”