Af3565

Histology fresh frozen sections were fixed in 4% PFA and stained followed by hematoxylin and eosin staining. For immunofluorescence, pancreatic tumours were fresh frozen in OCT, fixed in 4% PFA or acetone, and stained according to manufacturer's protocol. Sections were stained using the polyclonal Ki67 (Rabbit Anti-Ki67, Abcam ab15580), Trypsin 3/PRSS3 (Goat anti-Trypsin, R&D Systems AF3565) and MECA-32 (Rat anti-PLVAP, in-house hybridoma supernatant). For immunofluorescence the following detection antibody were used: Donkey anti-Rabbit 488 (Life Technologies), Donkey anti-Goat Alexa Fluor 546 (Life Technologies), Donkey anti-Rat 488 (Life Technologies) and DAPI (Life Technologies). Images were acquired using a Ziess LSM 510 meta confocal microscope.

Frozen sections of pancreatic tissue were incubated with a rabbit anti-Piezo1 (Alomone; #APC-087; 1:300) and/or a mouse anti-trypsin 3 (R&D; AF3565; 1:400) antibody. The signal from Piezo1 immunostaining was amplified by tyramide signal amplification following the manufacturer’s recommendations (Life Technologies; #T20924). All images were taken with a Zeiss 780 confocal microscope with either a ×20 objective or a ×63 oil-immersion objective.