Duo92002

Cells on coverslips were fixed using 4% paraformaldehyde (Electron Microscopy Sciences #15710). Cell membranes were permeabilized using 0.2% Triton X-100 (Thermo Fisher Scientific #9002–93-1). Cells were immuno-stained with anti-V5 and anti-Flag antibodies followed by duolink assay (DUO92008-3, DUO92004, DUO92002, Sigma-Aldrich) according to the manufacturer’s instructions. Coverslips were mounted with VectaShield/DAPI (H-1200, Vector Laboratories) on microscopy slides and analyzed using Olympus confocal microscope and Olympus Fluoview FV1000 software (https://www.olympus-lifescience.com/en/downloads/detail-iframe/?0[downloads][id]=847249651).

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).

Glass coverslips were placed in each well of 12-well plates and coated with type I collagen, and a culture insert was firmly placed on the coverslip. Keratinocytes were isolated from P0–P2 pups, plated within a culture insert at 3.2 × 10⁵ cells per insert and cultured in mKER medium for 2 d. Upon reaching confluence, culture inserts were removed to trigger cell migration. After 1 d, cells were fixed with 4% PFA at room temperature for 15 min, permeabilized with 0.1% Triton at room temperature for 10 min, and blocked with 2.5% donkey serum in PBS at room temperature for 1 h. Fixed cells were incubated overnight at 4°C with mouse anti–myosin IIA and rabbit anti-K6a primary antibodies (see above). Donkey anti-mouse secondary antibody conjugated to oligonucleotide (minus; DUO92004; Sigma-Aldrich) and donkey anti-rabbit secondary antibody conjugated to oligonucleotide (plus; DUO92002; Sigma-Aldrich) were added to the cells and incubated for 1 h at 37°C. The ligation and amplification steps were performed according to the manufacturer’s instructions (Duolink PLA; Sigma-Aldrich). Fluorescence was detected using an AxioObserver Z1 microscope equipped with an EC Plan Neofluar 40×/1.30 oil DIC M27 objective, an Apotome attachment, and Zen software. All images in a given experiment were taken under identical imaging parameters.