Topical ROL stimulates TGF-β/CTGF pathway, the major regulator of ECM homeostasis, in aged human skin in vivo. OCT-embedded skin sections (7μm) were obtained from aged (76±6 years) healthy sun-protected buttock skin after topical treatment of vehicle and 0.4% retinol for seven days. (A) HSP47 immunostaining. Representative images of twelve individuals (N=12). Bars=100μm. (B) TGF-β pathway components mRNA levels. Total RNA was prepared from human skin samples. mRNA levels were determined by real-time RT-PCR. mRNA levels were normalized to 36B4 (internal housekeeping gene control). Mean ± SEM, N=12, *p<0.05. (C) TGF-β1 (D) CTGF/CCN2 in situ hybridization. Representative images of twelve individuals (N=12). Bars= 100μm. (E) TGF-β1 (F) CTGF/CCN2 Northern analysis. The intensities were quantified and normalized using 36B4 as loading control. Insets show representative Northern blots. Data are expressed as mean±SEM, *p<0.05. N=12. (G) TGF-β1 (H) CTGF/CCN2 immunostaining. Representative images of twelve individuals (N=12). Bars= 100μm. (I) Smad7 Northern analysis. The intensities were quantified and normalized using 36B4 as loading control. Inset shows representative Northern blots. Data are expressed as mean±SEM, *p<0.05. N=12. (J) Smad7 protein. Smad7 protein levels were determined by Western analysis. Protein levels were normalized to β-actin (loading control). Insets show representative Western blots. Mean ± SEM, N =6, *p < 0.05. All positive staining was quantified by computerized image analysis (Image-pro Plus software, version 4.1, Media Cybernetics, MD). Data are expressed as mean±SEM, *p<0.05. N=12.