Aperio scanscope console 12

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).

IHC was performed as follows: all tissues were paraffin-embedded and cut into 4μm thick sections. All sections were dewaxed with xylene and hydrated with alcohol. Sodium citrate was used for antigen retrieval, and 0.3% of hydrogen peroxide (H₂O₂) was used to block endogenous peroxidase. After blocking non-specific sites with bovine serum albumin, all the sections were incubated with an appropriate primary and secondary antibody. We used the 3,3-diaminobenzidine (DAB) kit (8801-4965-72, Thermo Fisher) for visualization, and hematoxylin was used to stain nuclei. All the sections were dehydrated with alcohol and sealed with neutral resin. The IHC staining score was calculated based on pixel intensity as follows: no staining, 1; weak staining, 2; moderate staining, 3; strong staining, 4. Pathological images were acquired using Leica Aperio ScanScope Console 12.3.