Demonstration of apoptotic KCs in AD, atopy patch tests, ACD, and in vitro induced eczematous dermatitis. Skin sections were subjected to TUNEL (a, b, e–g) or HOECHST staining (c, d) without counterstaining. (a) Healthy normal skin as a negative control (left); lesional skin of acute AD (right). Red condensed and partly fragmented nuclei indicate positive staining of apoptotic KCs (some of these are indicated by arrows). ×400. (b) Left: Acute ACD; 2+ patch test 48 hours after exposure to 5% nickel sulfate. Right: 2+ atopy patch test 72 hours after exposure to 10,000 protein nitrogen units of house dust mite. ×400. (c) Normal, healthy skin as a negative control. ×200. (d) Left: Lesional skin of acute AD. Right: Acute ACD. 2+ patch test 48 hours after exposure to 5% nickel sulfate. Apoptotic cells show condensed or fragmented nuclei (some of these are indicated by arrows). ×400. (e) Normal human skin after 3 days of in vitro culture with unstimulated CD45RO⁺ T cells. Left: No TUNEL-stained KCs are detectable. Right: Normal human skin after 3 days of in vitro culture exposed to stimulated CD45RO⁺ T cells. Features of spongiosis and numerous apoptotic KCs are detectable (arrows). ×200. (f) Cultured skin equivalent after 3 weeks of in vitro culture. Left: KC layers and the stratum corneum are visible (hematoxylin/eosin staining). Right: TUNEL staining. No TUNEL-stained nuclei are detectable. ×400. (g) Cultured skin equivalent after 3 days exposed to stimulated CD45RO⁺ T cells. There are signs of KC damage (left). With the TUNEL technique, several apoptotic nuclei are stained in the basal and suprabasal epidermis (arrows, right). Results shown in e–g are representative of two experiments with healthy skin and three experiments with cultured skin equivalents.