Detecting Fibronectin mRNA Expression

Digoxigenin-11-dUTP single-stranded RNA probes for detecting fibronectin mRNA were prepared using a digoxigenin RNA labeling kit (Roche). The sections were treated with proteinase K and acetic anhydride and overlaid with 150 μl of hybridization solution containing the digoxigenin-labeled fibronectin probe (1 μg/ml). Then, they were denatured at 70°C for 60 minutes and hybridized overnight at 65°C. Hybrids were detected with an anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche). Sections were incubated in 1% bovine serum albumin/PBS for 1 hour before incubation with the primary antibody. Primary antibodies specific for fibronectin (kindly provided by K. Yamada), collagen IV [1 (link)], dentin sialoprotein (DSP) [20 (link)], ameloblastin [20 (link)], laminin β1γ1 (MAB1905; Merck Millipore), and β1 integrin (9EG7: BD Biosciences) were detected using Alexa488- or Alexa594-conjugated secondary antibodies (Invitrogen). Nuclei were stained with Hoechst dye (Sigma-Aldrich) or DAPI (Vector Laboratories). A fluorescence microscope (BZ-8000, KEYENCE) was used for imaging analysis. Images were prepared using a BZ analyzer (KEYENCE) and Adobe Photoshop (Adobe Systems, Inc.).