Alexa flour 568 conjugated

Ex vivo tumour samples preserved in 4% paraformaldehyde were then dehydrated in 40% sucrose solution at 4 °C. Tumour tissues were embedded in o.c.t compound (VWR, Leicestershire, UK) and cryosectioned into 25-μm-thick sections. Frozen sections were blocked in 5% normal donkey serum at room temperature for 1 h and then incubated overnight at 4 °C with one of the primary antibodies: mouse monoclonal HGF (Santa Cruz Biotechnology, TX, USA), mouse monoclonal TIMP-2 (Santa Cruz Biotechnology), mouse monoclonal CD68 antibody (Santa Cruz Biotechnology), rat monoclonal Ly6g antibody (Abcam plc, Cambridge, UK), rabbit monoclonal CD11c antibody (Cell Signalling technology, London, UK) or mouse CD54 antibody (Novus biologicals, Abingdon, UK). Slides were then incubated the following day with Alexa flour 568-conjugated (ThermoFisher scientific) or Alexa flour 488-conjugated (Abcam plc) secondary antibody incubation. Slides were counterstained with Vectashield mounting medium containing DAPI (Millipore, Watfield, UK). Slides were viewed under Olympus BX4 microscope (Olympus, Hamburg, Germany).

Antibodies and chemicals used in the present study are listed as follows. Anti (α)-PRRSV-nsp1β PAb (rabbit polyclonal antibody) specific for PRRSV-2 nsp1β was generated at the Immunological Research Center, University of Illinois at Urbana-Champaign (Urbana, IL). α-N protein MAb (MR40; mouse monoclonal antibody) was obtained from E. Nelson (South Dakota State University, Brookings, SD). α-p65 Mab (mouse) (F-6, sc-8008) was purchased from Santa Cruz Biotechnologies Inc. (Santa Cruz, CA). Alexa-Flour 488-conjugated and Alexa-Flour 568-conjugated secondary antibodies were obtained from ThermoFisher (Rockford, IL). Human tumor necrosis factor-α (TNF-α) (8902) was purchased from Cell Signaling (Danvers, MA). DAPI (4′, 6′-diamidino-2-phenylindol) and Polyinosinic:polycytidylic [poly (I:C)] were obtained from Sigma (St. Louis, MO). Human and porcine recombinant IFN-β were purchased from Calbiochem (San Diego, CA), and for stimulation, 1000 unit/ml was added to cells for 6 h.

For immunofluorescence staining, we used antibodies against rhodamine-phalloidin (1:200, Yeasen, #40734ES75), C-terminal binding protein-2 (CtBP2, 1:400, mouse IgG1, BD Bioscience, #612044¹), glutamate receptor 2 (GluR2, 1:200, mouse IgG2a, Millipore, Cat #mab397²), and 4′, 6-diamidino-2-phenylindole (1:1,000, Sigma, United States, #D9542).
Whole-mount organs of Corti were permeabilized with 0.1% Triton X-100 for 40 min and then blocked with 10% normal goat serum for 30 min at 37°C. Subsequently, the tissue was incubated with the primary antibodies overnight at 4°C. After rinsing, the samples were incubated with their specific secondary antibodies: 1:500 of goat anti-mouse IgG1 (Invitrogen, Alexa Flour™ 568-conjugated, ref#A21134, RRID: AB_2535766) and goat anti-mouse IgG2a (Invitrogen, Alexa Flour™ 647-conjugated, ref#A21241, RRID: AB_2535810) at 37°C for 2 h. The samples were imaged using a laser confocal microscope (Leica SP8, Germany).