The Localization of Label-Retaining Cells in Mouse Nails Motonobu Nakamura, Osamu Ishikawa  Journal of Investigative Dermatology  Volume 128, Issue 3, Pages 728-730 (March 2008) DOI: 10.1038/sj.jid.5701062 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Label-retaining cells in the mouse nails. (a) Label-retaining cells in the nails (arrows). Five newborn BALB/c Cr Slc mice were purchased from the SLC company (Hamamatsu, Japan) and maintained at the Institute of Laboratory Animals, Gunma University. BrdU (100mg/kg/day; Sigma-Aldrich, St Louis, MO) was injected subcutaneously daily for 5 days. Mice were killed after 4 weeks and frozen nail sections were stained with a BrdU IHC kit (Kamiya Biochemical Company, Seattle, WA) according to the manufacturer's instructions. (b) Low-magnification photomicrograph of the sagittal section of the nails. The proximal, middle, and distal aspects of the distal phalanx of one digit in Figure 2d and e. (c) Lhx2 expression in the nail matrix cells (arrows). Frozen sections of 5-week–old female BALB/c Cr Slc mouse nail were stained using 100 times diluted anti-Lhx2 antibody (sc-19342; Santa Cruz Biotechnology, Santa Cruz, CA) and LSAB+System-HRP (Dako Cytomation, Glostrup, Denmark) according to the manufacturer's manuals. Bar=100μm. Journal of Investigative Dermatology 2008 128, 728-730DOI: (10.1038/sj.jid.5701062) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Expression of Rspo4, Fzd8, and Lrp6 mRNA in the mouse nails. (a) Expression of Rspo4 mRNA in E18 and 6-week-old mouse digits. Total RNA was extracted from all digits with columns (n=4, RNeasy Mini kit; Qiagen, Hilden, Germany) and was reverse transcribed into cDNA with first-strand cDNA synthesis kit RT–PCR (Roche Diagnostics, Indianapolis, IN). The expression levels of Rspo4 and glyceraldehyde-3-phosphate dehydrogenase were examined using 20 × Assays-on-Demand Gene Expression Assay Mix (Mm00615419_m1 and Mm_99999915_g1, respectively; (Applied Biosystems, Foster City, CA)) with 7300 system (Applied Biosystems), according to the manufacturer's instructions. A P-value lower than 0.05, indicated by an asterisk, was considered significant (n=4, mean±SD). (b, c) Expression of Fzd8 (b) and Lrp6 (c) protein in the nails (white arrows). Paraffin sections of 5-week–old female BALB/c Cr Slc mouse nails were stained using 100 times diluted anti-Fzd8 antibody (sc-33505; Santa Cruz Biotechnology), anti-Lrp6 antibody (sc-17984; Santa Cruz Biotechnology), and LSAB+System-HRP (Dako Cytomation), according to the manufacturer's instructions. (d, e) Expression of Fzd8 (d) and Lrp6 (e) mRNA in the nails. BALB/c Cr Slc female 4-week-old mice were purchased from SLC. We sampled the distal phalanx and cut it into three parts, the proximal, middle, and distal portions (Figure 1b). Distal nail refers to the hyponychium and parts of the nail bed; middle refers to parts of the nail bed and the matrix; and proximal refers to the matrix and the nail fold epithelium along with all the underlying connective tissue. Total RNA was extracted from the nails with columns (n=4; RNeasy Mini Kit; Qiagen) and was reverse transcribed into cDNA by first-strand cDNA synthesis kit RT–PCR (Roche Diagnostics). The expression levels of Fzd8, Lrp6, and glyceraldehyde-3-phosphate dehydrogenase were examined using 20 × Assays-on-Demand Gene Expression Assay Mix (Mm00433419_s1, Mm00521783_m1, and Mm_99999915_g1, respectively; Applied Biosystems) with 7300 system (Applied Biosystems) according to the manufacturer's instructions. We used the t-test to compare the expression level of Fzd8 and Lrp6. A P-value lower than 0.05, indicated by an asterisk, was considered significant (n=4, mean±SD). The same difference was confirmed using actin beta (Actb) gene expression (Mm00607939_s1) as a control (data not shown). Bar=100μm. Journal of Investigative Dermatology 2008 128, 728-730DOI: (10.1038/sj.jid.5701062) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions