Sample preparations of total RNA for qRT-PCR and protein isolation for Western Blot analyses were performed as described previously [10 (link)] To measure the levels of Foxn1, Mmp-9, Mmp-13, Hprt1 mRNA expression, single tube TaqMan® Gene Expression Assays (Life Technologies) were used. Each run included a standard curve based on aliquots of RNA pooled from different skin tissue samples. All samples were analyzed in duplicates. mRNA expression levels were normalized to the reference gene - Hprt1 (hypoxanthine phosphoribosyl transferase 1) and multiplied by 10.
Primary antibodies used in Western Blot analysis were rabbit anti-involucrin (Covance; Princeton, NJ, USA), rabbit anti-Mmp-9 (Millipore, Billerica, MA, USA) and mouse anti-Gapdh (AbCam, Cambridge, UK). As secondary antibodies IRDye800TM and Cy5.5 (Rockland, Limerick, PA, USA) were used. Bands were visualized using the Odyssey imaging system (LI-COR Bioscience) according to the manufacturer’s protocol. Densitometric protein analysis was performed as previously described [12 (link)].
Primary antibodies used in Western Blot analysis were rabbit anti-involucrin (Covance; Princeton, NJ, USA), rabbit anti-Mmp-9 (Millipore, Billerica, MA, USA) and mouse anti-Gapdh (AbCam, Cambridge, UK). As secondary antibodies IRDye800TM and Cy5.5 (Rockland, Limerick, PA, USA) were used. Bands were visualized using the Odyssey imaging system (LI-COR Bioscience) according to the manufacturer’s protocol. Densitometric protein analysis was performed as previously described [12 (link)].
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