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 [3H]-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 degrees 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.