PMC:7376845 / 8883-9946 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7376845","sourcedb":"PMC","sourceid":"7376845","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7376845","text":"Transient transfection and immunofluorescence analysis\nFor immunofluorescence (IF) analysis, COS-7 cells on glass coverslips were transfected as above and fixed at 24 hours post-transfection in 4% paraformaldehyde for 10 min at RT followed by permeabilisation with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States) for 5 min. Fixed cells were then blocked with PBS containing 10% FBS for 30 min at RT. Cells were immunolabelled for 1 hour at RT with the indicated murine mAb and 45 min with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Life Technologies, Carlsbad, CA, United States). Immunolabelled coverslips were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and mounted using ProLong Gold Antifade Mountant (Molecular Probes, Eugene, OR, United States). Images were acquired with Olympus CKX53 microscope using Olympus (Tokyo, Japan) LCAch N 20×/0.40 iPC objective lens and Olympus DP27 colour camera with Olympus cellSens software. Each channel was collected separately, with images at 1024 × 1024 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