Anti f4 80 bm8

Tissue samples were cut and snap frozen in optimum cutting temperature (O.C.T., Fisher Scientific, Houston, TX). 6 um cryo-sections of tissue sections were cut and fixed with pre-cold acetone for 20 min. Immunostaining was performed as previously described (Fu et al., 2014 (link)). Before adding primary mAbs, sections were blocked with 5% normal donkey serum (Jackson ImmunoResearch, West Grove, PA). The following mAbs were used in different combinations as indicated in the figure legends: anti-CRIg mAb (a gift from Genentech, South San Francisco, CA); anti-F4/80 (BM8, BioLegend); anti-CD4 (RM4-5, BioLegend). Nuclei were stained with DAPI (4',6-Diamidino-2–28 phenylindole dihydrochloride) (Invitrogen). Images were acquired on an AxioImager microscope (Zeiss, Thornwood, NY), and were processed with ImageJ (NIH). For histology assays, the pancreases were removed and fixed with 10% formalin solution. Fixed tissue blocks were paraffin-embedded, sectioned, and stained with haematoxylin and eosin.

Excised tumors were washed with PBS, dissected into smaller fragments using scalpels, and further dissociated into single cell suspensions using the Miltenyi Tumor Dissociation Kit (#130-096-730) and the GentleMACS Octo dissociator (Miltenyi #130-095-937). The digested tumors were filtered through 100μM & 30μM pre-separation filters (Miltenyi #130-098-463,130-098-458), washed with PBS, and then used for flow cytometry or RNA based analysis.
For each sample, 1 x 10⁶ cells were treated with mouse Fc Blocking solution (Miltenyi, #130-092-575) and then stained with a defined panel containing live/dead stain (Life Technologies, #L34957) and seven different labeling antibodies. Anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53–6.7), anti-CD45R (RA3-6B2), anti-CD25 (PC61), anti-CD11b (M1/70), anti-CD11c (HL3) were purchased from BD Biosciences. Anti-CD14 (Sa2-8), anti-FoxP3 (FJK-16s), and anti-CD45 (30-F11) were purchased from eBioscience. Anti-CD49b (DX5), anti-GR1 (RB6-8C5), and anti-F4/80 (BM8) were purchased from BioLegend. Perm/Wash Buffer (BD Biosciences, #554714) was used to permeabilize and facilitate intracellular staining. All samples were fixed with 2% paraformaldehyde, acquired on a BD FACSCanto II, and analyzed by FlowJo software (v10.1r7).

BrdU was delivered to mice through drinking water at the concentration of 1mg/ml + 1% glucose. Spleen and lymph nodes were harvested on day 6 post-immunization or infection. Following tetramer enrichment, cells were stained with cell surface antibodies [anti-CD3, anti-CD4, anti-CD19 (eBio (1D3), eBiosciences), anti-CD8α, anti-CD11c (eBiosciences), anti-F4/80 (BM8, Biolegend) and anti-CD5 (53–7.3, BD Pharmingen)] followed by intracellular anti-BrdU (BD Pharmingen) antibody according to the BrdU flow kit protocol (BD Biosciences).