Sirius red

Alcian blue and Sirius red staining of mouse pancreas tissues were carried out according to published protocols.^{36 (link)} The expression of mucin was stained with Alcian blue reagents (IHC WORLD, Woodstock, USA, IW3000) according to manufacturer’s instructions. Briefly, pancreas sections were deparaffinized, hydrated, and immersed in Alcian blue solution for 30 min at room temperature. After washing with distilled water, the slides were counterstained in nuclear fast red or eosin. Strongly acidic mucosubstances will be stained blue. The presence of collagen was stained with Sirius red (IHC WORLD, Woodstock, USA, IW3012). In brief, slides were deparaffinized and hydrated and nuclei were stained with Weigert’s hematoxylin for 10 min. Following 10 min of washing in running tap water, the slides were stained in picro-Sirius red solution containing 0.1% Sirius red in saturated aqueous solution of picric acid for 1 h. Collagen-rich tissue is stained red on a pale-yellow background.

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.