In situ hybridization analysis of Tmprss6 and BMP6 mRNA in mouse liver tissue. 10μM thick liver tissue sections from a 10-week-old 129/SvEvTac mouse were probed with digoxigenin-labeled antisense riboprobes specific for mouse Tmprss6 (A) or mouse BMP6 (B), respectively. Sense probes were used as negative controls. In panel B, images at the bottom (* and **) are the enlargement of the boxed portions (* and **) in the corresponding top panels. Digoxigenin-labeled antisense and sense riboprobes for mouse BMP6 and Tmprss6 were synthesized by in vitro transcription by the use of either MEGAscript SP6 kit or MEGAscript T7 kit (Ambion). The fragments of mouse BMP6 and Tmprss6 cDNA used for riboprobe synthesis were amplified from a mouse liver cDNA preparation by PCR by use of the Expand High-Fidelity PCR system (Roche Applied Science), followed by cloning into pGEM-T vector (Promega). The primers used for amplifications are listed in Table 2. The amplicons were confirmed by DNA sequencing. Images were taken under × 100 original magnification. In situ hybridization analyses for both Tmprss6 and BMP6 mRNA were performed at least 3 times with 2 different sets of probe preparations and showed consistent results. Scale bar, 50 μm.