Ccl15

Human pancreatic tissue slides, including normal and cancer tissue samples, were obtained from BioChain (Newark, CA, USA) and US Biomax (Derwood, MD, USA). All experiments that involved using human tissue samples were carried out according to the IRB protocol approved by the CAU IRB committee. Slides were deparaffinized in xylene and gradually re-hydrated through 100% alcohol to distilled water. The re-hydrated slides were subjected to heat-induced antigen retrieval in the antigen retrieval buffer, 10 mM sodium citrate buffer (pH 6.0) or 10 mM Tris. Slides were then incubated with 3% hydrogen peroxide followed by PBS wash. Slides were treated with protein block serum-free solution (DAKO) for 10 min at room temperature. After the primary antibody (CCL15 1:400 (Abcam); CCR1 1:100 (Novus Biologicals); CCR3 1: 200 (Abcam)) was incubated with slides, the ImmPRESS Polymer Detection Kit (Vector Laboratories; Burlingame, CA, USA) was used according to the manufacturer’s instructions. Images were collected using the Aperio VERSA tissue scanner with ImageScope software (Aperio; Sausalito, CA, USA). For specificity of CCR1 and CCR3 antibody, see Figure S10.

CCL9, CCL15, CCR3, MMP14 and CD3 antibodies were purchased from Abcam (Cambridge, UK). Both F4/80 and Ly6B.2 antibodies were from Bio-Rad (Hercules, CA, USA). CCR1 antibody was purchased from Novus Biologicals (Littleton, CO, USA). MMP2 and MMP3 antibodies were obtained from Thermo Fisher Scientific (Walthem, MA, USA). In addition, MMP3 antibody was also purchased from ProteinTech (San Diego, CA, USA). Recombinant CCL9 was from PeproTech (Cranbury, NJ, USA). CCL9 neutralizing antibody and its isotype control antibody were purchase from R&D Systems (Minneapolis, MN, USA) and BioLegend (San Diego, CA, USA), respectively. N-Acetyl Cysteine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Other reagents used are described in the specific experiment sections.