In Vivo Labeling and Analysis of Epidermal Stem Cells

To identify slowly cycling (label-retaining) keratinocytes in the cutaneous epithelium of mice, newborn female pups were injected with [³ H]-thymidine twice daily for 3 d beginning the third day after birth. At 8–10 wk of age, skin from the dorsal side of the mice was removed and fixed. The skin was processed for paraffin embedding, and serial sections were prepared. Dewaxed and ethanol-treated slides were dried and dipped into autoradiographic emulsion and allowed to dry in light tight box for 4 wk at 4�C. After fixation with photographic fixer, the slides were washed, lightly stained, and viewed with a light microscope to observe and quantify cells that retained the labeling. Label-retaining cells were identified by the black silver grains over their nuclei.