F4 80 ab

After 31 days of growth, tumor volumes in the PD-1 Ab group had reached the maximum allowed size according to the IACUC approved protocol. Mice were ethically euthanized by CO₂ asphyxiation and cervical dislocation and were weighed. Pancreatic tumors were excised, weighed, and fixed in 4% paraffin formaldehyde. Eight μm cuts of paraffin-embedded tumors were mounted on slides for staining. Tumor-associated fibrosis was revealed with Masson’s trichrome stain. Images from each slide were captured using a 20X objective lens on an Olympus BX61 microscope with a DP73 camera (N=10 per group). Analysis of Masson’s trichrome was done using software by Image-J by two investigators blinded to treatment of the area of fibrosis.
For immunohistochemistry analysis of TILs, 5 μm fixed tumor sections were stained with either CD8 Ab (1:75; eBioscience); or Foxp3 Ab (1:30; eBioscience) and immunoreactive cells were counted manually. Tumor-associated macrophages (TAMs) were reacted with an F4/80 Ab (1:40; eBioscience) and images were taken on an Olympus BX61 microscope with a DP73 camera. The number of immunoreactive cells per slide, were counted with image-J computer software by investigators blinded to the treatments.

Five μm cuts of paraffin-embedded liver sections from mice fed CDE diet with untreated water (N= 15) and mice fed CDE diet with proglumide-treated water (N=15) were mounted on slides. Slides were reacted with an F4/80 Ab (1:40) at 4°C overnight; (eBioscience; Cat # 14–4801-85) followed by incubation with a secondary M.O.M antibody (ImmPRESS; Cat# MP-2400) for 30 minutes at room temperature. After counterstaining with hematoxylin (1:9) the number of immunoreactive cells per slide, were counted with ImageJ computer software.