3 3 diaminobenzidine tetrahydrochloride plus dab

Western blot analysis was performed as previously described (124 (link)). Human pancreatic TMAs (#PA1001 and #PA207) were purchased from US Biomax Inc. Mouse pancreas was fixed in 10% buffered formalin and embedded in paraffin. For IHC, unstained pancreatic slides were deparaffinized and heated in standard citrate or tris-EDTA retrieval buffer for 30 min at 95°C After incubation overnight with the primary antibodies at 4°C, the slides were incubated with biotinylated secondary antibodies (Vector Laboratories Ltd.) for 1 hour at room temperature. Antibody labeling was visualized using the VECTASTAIN ABC kit (Vector Laboratories Ltd.) followed by staining with 3,3′-diaminobenzidine tetrahydrochloride plus (DAB+) according to the manufacturer’s instructions (Thermo Scientific) or using the Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories Ltd.). The antibodies used for Western blot and IHC analyses are listed in table S5. Collagen staining was performed using the Masson Trichrome Stain Kit according to the manufacturer’s instructions (Sigma).

Dissected tissues were fixed, immediately after removal, in a 10% buffered formalin solution for a maximum of 24 hr at room temperature before being dehydrated and paraffin-embedded under vacuum conditions. Seven-micrometer sections were deparaffinized in xylene, hydrated in graded alcohols and heated in standard citrate or Tris-EDTA retrieval buffer for 30 min at 95°C. After incubation overnight with the primary antibodies at 4°C, the slides were incubated with biotinylated secondary antibodies (Vector Laboratories Ltd.) for 1 hour at room temperature. Antibody labeling was visualized using the VECTASTAIN ABC kit (Vector Laboratories Ltd.) followed by staining with 3,3’-diaminobenzidine tetrahydrochloride plus (DAB+) according to the manufacturer’s instructions (Thermo Scientific) or using the Vector Blue Alkaline Phosphatase Substrate Kit (Vector Laboratories Ltd.).