The Measurement of Double Staining for Senescence Associated-beta-Galactosidase Activity and Keratin 10 or Involucrin in Monolayer and Organotypic Cultured Keratinocytes

Kim, Jong Whee; Liu, Yin; Sung, Hoon Ki; Jung, Tae Eun; Park, Jeong Hyun; Kim, Joo Young; Song, In Hwan; Lee, Yung Chang; Sung, Eon Gi

Abstract

This experiment developed the methodology of double staining for senescence associated-beta-galactosidase (SA-beta-gal) activity and keratin 10 (K10) or involucrin. To prove the usefulness of the double staining, the author investigated the relationship between senescence and differentiation in monolayer and organotypic cultured keratinocytes. The results were as follows: K10 and involcrin together with SA-beta-gal were doubly stained in most of monolayer cultured keratinocyte. This fact indicated that the senescence and differentiation had simultaneously occurred in the same keratinocyte. In spite of the advantages to preserving structures, the paraffin specimen was not suitable for double staining because of the limitation of SA-beta-gal reactivity. Although the cryosectioned specimen did not have the morphology as good as the paraffin specimen, it was suitable for double staining due to the goodness of SA-beta-gal reactivity. Double staining well reflected the disturbances of senescence and differentiation which could be caused by deranged organizations of the organotypic cultured skin. The organotypic cultured skin which showed deranged organizations such as stratified basal layer, no typical cell features in each epdermal layer, and wide intercellular spaces had SA-beta-gal activity in epidermis and K10 or involucrin reaction in basal cell. But the skin which showed well arranged organizations resembling in vivo skin had no SA-beta-gal activity and no K10 or involucrin reaction in basal cells. In conclusion, it might be suggested that the double staining for SA-beta-gal activity and K10 or involucrin could be used for detecting the extent of senescence and differentiation in the same cell.