Dapi containing media

Twenty cases of PDAC liver metastases and adjacent livers tissues were obtained from Ren Ji hospital from January 2015 to June 2018 (Supplementary Table 1). The study was conducted in accordance with International Ethical Guidelines for Biomedical Research Involving Human Subjects (CIOMS). The study was approved by the Research Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. All patients had not received radiotherapy, chemotherapy, hormone therapy or other related antitumor therapies before surgery. Written informed consent was provided before enrollment. Hematoxylin and eosin (H&E) staining was performed routinely and observed with Leica Aperio ScanScope Console 12.3. For immunofluorescence staining, paraffin sections were dewaxed with gradient ethanol, steam heated for antigen retrieval in citrate-based buffer, blocked with PBS containing 10%BSA for 1 h, stained with anti-P2RX1 (Alomone, 1:200), and CD66b (Biologend, 1:1000), Ly6G (Biolegend, 1:1000), or citrullinated histone H3 (CitH3) (Alomone, 1:200) overnight and secondary antibody (Abcam, 1:1000) for 2 hr and finally mounted in DAPI-containing media (Vector Labs) for imaging on a laser scanning confocal microscope (Leica SP8 LAS X).

Adult female flies were dissected in PBS. All the digestive tract was removed and fixed in PBS and 4% electron microscopy grade paraformaldehyde (Polysciences, USA) for 40 minutes. Samples were rinsed 3 times with PBS, 4% BSA, 0.1% Triton X-100 (PBT-BSA), incubated with the primary antibody overnight at 4 °C and with the secondary antibody for 2 hours at room temperature. Finally, the samples were rinsed 3 times with PBT-BSA and mounted in DAPI-containing media (Vectashield, USA). All the steps were performed without mechanical agitation. Primary antibody mouse α-Pros (1:100) was obtained from the Developmental Studies Hybridoma Bank (DSHB). Secondary antibodies were from Invitrogen (USA). Images were obtained on a Leica SPE or Leica SP5 confocal microscopy and processed in Photoshop CS5 (Adobe, USA).

Adult female flies were dissected in PBS. All the digestive tract was removed and fixed in PBS and 4% electron microscopy grade paraformaldehyde (Polysciences, USA) for 35 minutes. Samples were rinsed 3 times with PBS, 4% BSA, 0.1% Triton X-100 (PBT-BSA), incubated with the primary antibody overnight at 4°C and with the secondary antibody for 2 hours at room temperature. Finally, the samples were rinsed 3 times with PBT-BSA and mounted in DAPI-containing media (Vectashield, USA). All the steps were performed without mechanical agitation. Primary antibodies mouse α-Pros (1:100), α-Dl (1:10), α-Arm (1:10), α-Dlg (1:250) and α-Sn (1:50) were obtained from the Developmental Studies Hybridoma Bank (DSHB), rabbit α-PH 3 (1:100) from Cell Signalling (USA), rabbit α-Pdm1 (1:1000) was a gift of Dr.Yang Xiaohang (Institute of Molecular and Cell Biology, Singapore), rabbit α-laminin (1:1000) was from Sigma (USA) and goat α-GFP (1:500) was form Abcam (UK). Secondary antibodies were from Invitrogen (USA). TRICT-conjugated Phalloidin (Sigma, USA) was used at 5 µg/ml. Images were obtained on a Leica SPE or Leica SP5 confocal microscopy and processed in Photoshop CS5 (Adobe, USA).