Nuclear red

Paraffin embedded sections were analyzed by TUNEL assay (DermaTACS, Trevigen, Gaithersburg MD) counterstained with Nuclear Red (Vector Laboratories, Burlingame CA). Cell death was quantitated by photographing four consecutive fields. The percent of apoptotic cells was determined by counting the number of epidermal blue apoptotic cells and dividing by the total number of epidermal cells in the field. In addition to the histologic assessment described above, we quantitatively evaluted apoptosis by measuring caspase-3 activity in the supernatant of homogenized tissue samples using the luminescence Caspase- GloR 3/7 assay kit (Promega, Madison, WI). Three 6 mm punch biopsies were homogenized in 300 μl of PBS+2mM EDTA and were centrifuged at 1000 g for 10 min. Results were normalized to mg tissue for mouse skin and mg protein for human skin.

Epidermal cells containing pyknotic nuclei and eosinophilic cytoplasm were counted in H&E stained sections. To confirm the presence of epidermal apoptotic cells, paraffin embedded sections were analyzed by TUNEL assay (DermaTACS, Trevigen, Gaithersburg MD) and counterstained with Nuclear Red (Vector Laboratories, Burlingame CA). The percent of sunburned/apoptotic cells was quantitated by counting five fields at 400X and averaging results to obtain a single value per sample.