Alcian blue

For H&E staining, slides were exposed to hematoxylin for 7 minutes (VitroVivo), then immersed in 95% alcohol for 1 minute, followed by eosin staining for 30 seconds. H&E-stained slides were analyzed for PanINs and adenocarcinoma under masked conditions by a board-certified pathologist. Tissues were also stained with (i) Alcian blue (IHC World) followed by counterstaining with Nuclear Fast Red (Vector Labs) and (ii) Sirius red (IHC World) followed by counterstaining with hematoxylin. Staining was quantified using a stereological approach. A grid was placed over an image of the entire pancreas, and the intersection points landing on tissues positively stained for either Alcian blue or Sirius red were counted. To obtain the stereologic score, the number of intersection points landing on positively stained tissues was divided by the total number of intersection points counted for the slide.

For H&E staining, slides were exposed to hematoxylin for 7 minutes (VitroVivo), then immersed in 95% alcohol for 1 minute, followed by eosin staining for 30 seconds. H&E-stained slides were analyzed for PanINs and adenocarcinoma under masked conditions by a board-certified pathologist. Tissues were also stained with (i) Alcian blue (IHC World) followed by counterstaining with Nuclear Fast Red (Vector Labs) and (ii) Sirius red (IHC World) followed by counterstaining with hematoxylin. Staining was quantified using a stereological approach. A grid was placed over an image of the entire pancreas, and the intersection points landing on tissues positively stained for either Alcian blue or Sirius red were counted. To obtain the stereologic score, the number of intersection points landing on positively stained tissues was divided by the total number of intersection points counted for the slide.

For adipogenic and osteogenic differentiation, cells were seeded on to six-well plates that contain differentiation medium. The composition of the differentiation medium is shown in Table 2. For chondrogenic differentiation, cells were cultured in 5 μl droplets of growth medium in four-well plates for 3 h in the presence of 5% CO₂ and changed with chondrogenic differentiation medium plus transforming growth factor β-3 (TGF-β3; Lonza, U.S.A.). All differentiation media were changed every 2–3 days and the differentiation to the three cell lineages was evaluated after 21 days.
To evaluate the differentiation abilities, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, and then washed with PBS again. For adipogenic differentiation, accumulation of red lipid vacuoles was observed after Oil Red O staining (IHC World, U.S.A.). For osteogenic differentiation, extracellular calcium deposition was confirmed by Alizarin Red staining (IHC World, U.S.A.). For chondrogenic differentiation, the presence of glycosaminoglycan was verified by Alcian Blue staining (IHC World, U.S.A.). Stained cells were visualized using an inverted microscope.