Tissue sections were processed and costained for FFAR2 and FFAR3 as previously described (29 (link)). Normal human colon sections were obtained from US Biomax (Rockville, MD, USA). HEK293 cells (control-HEK293, FFAR2-HEK293, FFAR3-HEK293, and FFAR2-FFAR3-HEK293), human monocytes, and macrophages were embedded in 1% agarose and fixed overnight with 10% neutral buffered formalin solution followed by embedding with paraffin with tissue processor. After antigen retrieval with 0.01 M citrate buffer pH 6, 20 min at 99°C, tissue sections were stained at 4°C overnight with primary antibodies against FFAR3 (clone 16F4.1; EMD Millipore, Billerica, MA, USA) or FFAR2 (sc-32906; Santa Cruz Biotechnology, Santa Cruz, CA, USA). For the proximity ligation assay (PLA), incubation with the PLA probes (Anti-Rabit Plus, DUO92002, and anti-mouse MINUS, DUO92004; both Sigma-Aldrich, St. Louis, MO, USA) followed by chromogenic substrate development and nuclear staining (Duolink In Situ Brightfield Kit, DUO92012; Sigma-Aldrich) was then performed. For immunohistochemical staining, incubation with horseradish peroxidase–polymer anti-mouse IgG (GBI Labs, Mukilteo, WA, USA) was followed by color development with liquid DAB+ substrate (GBI Labs).