Nbp1 53204

Anonymous paraffin-embedded human PDAC sections were obtained from Laboratory Pathology East Netherlands (LabPON; Hengelo, The Netherlands). Sections were deparaffinized in xylene and rehydrated in a serious of ethanol followed by MilliQ water. Antigen retrieval was performed by heat induction at 95°C in citrate buffer at pH 6.0 (Dako, Glostrup, Denmark). The sections were incubated with the primary antibody against alpha smooth muscle actin (αSMA, 1:500 dilution, 1A4, Sigma-Aldrich, St. Louis, MO, USA) and CK19 (1:100 dilution, NBP1-53204, Novus Biologicals, Littleton, CO, USA) in phosphate buffer saline (PBS) overnight at 4 °C. After washing in PBS, the sections were incubated with secondary fluorescent antibody (Alexa Fluor 488 donkey anti-rabbit IgG, 1:100 dilution, A27034 & Alexa Fluor 594 goat anti-mouse IgG, 1:100, A11032, Thermofisher Scientific, Waltham, MA, USA) for 30 min at room temperature, before being washed in PBS and mounted with Fluoroshield™ with DAPI (Sigma-Aldrich) and imaged using Nanozoomer-RS (Hamamatsu Photonics, Hamamatsu, Japan).

Panc-1/PSC µtissues and Panc-1/PSC heterospheroids were formed as previously described before being washed with DPBS, fixed with 4% formaldehyde (Sigma-Aldrich), washed again with DPBS and being embedded into Cryomatrix™ (Thermofisher Scientific) before being snap-frozen using isopentane (Sigma-Aldrich). Embedded and frozen µtissues and heterospheroids were cut into 6 µm thick cryosections, air-dried, and fixed with acetone for 10 min at room temperature. A circle was drawn around the tissues using a hydrophobic PAP pen (Sigma-Aldrich) before being rehydrated in PBS. The rehydrated sections were incubated overnight in primary antibody against CK19 (1:100 dilution, NBP1-53204, Novus Biologicals, Littleton, CO, USA) overnight at 4 °C. Next, the sections were washed with PBS, before being incubated with fluorescent secondary antibody (Alexa Fluor 594 donkey anti-rabbit IgG, 1:100 dilution, A-21207, Thermofisher Scientific), washed again in PBS and mounted using Fluoroshield™ with DAPI and imaged using Nanozoomer-RS.