Nb300 141

FFPE pancreata of C57BL/6 control mice at 2 and 10 months (n = 3/group) and KC mice at 1, 4, and 9 months (n = 3/group) were sectioned and subjected to in situ hybridization as previously described62 (link). FFPE pancreatic tissue sections (5 μm) from normal control and PDAC patients (n = 4/group) were also subjected to miR-29 in situ hybridization as described. Slides were hybridized with 50 nM 5′-biotin labeled U6 snRNA LNA probe (Exiqon, 99002-03) and 5′ and 3′ DIG labeled miR-29a LNA probe (Exiqon, 10000-899999-15) for 90 minutes at 55 °C. Subsequently, slides were probed with GFAP (Novus Biologic, NB300-141, 1:200) or CK19 antibody (Abcam, ab52625, 1:200) and stained with secondary antibodies conjugated to HRP to facilitate Tyramide signal amplification (TSA) reaction: goat anti-rabbit IgG (Santa Cruz, sc-2004, 1:500–1:1000), anti-Dioxigenin (Abcam, ab6212, 1:200), and Streptavadin (Thermo Scientific, #21130, 1:5000). Slides were then stained with Hoechst NucRed^TM Dead 647 (Life Technologies, #R37113) to stain nuclei. 20X images were taken using DeltaVision Core confocal microscope, projected in four channels, pseudo-colored, and merged. Corrected total cell fluorescence (CTCF) of miR-29a (green) was quantified in six or more randomly selected GFAP-positive PSCs/fibroblasts or CK19-positive epithelial cells using ImageJ analysis as previously described63 (link).