Dmi8 thunder imager fluorescent microscope

Formalin-fixed and paraffin-embedded lung sections from mock- and IAV-infected mice were prepared as previously described (52 (link)) and probed for epithelial or endothelial cell detection using monoclonal mouse anti–α-smooth muscle actin (αSMA) (1:10,000; A2547, Sigma-Aldrich, Darmstadt, Germany), rabbit anti-mouse CD31 (1:50; ab28364, Abcam, Cambridge, UK), or rabbit anti-mouse EpCAM (1:50; ab71916, Abcam, Cambridge, UK) antibodies. Tissues were incubated with corresponding secondary antibodies goat anti-mouse immunoglobulin G2a (IgG2a)–AF488 (1:200; A-21131, Thermo Fisher Scientific) and anti-rabbit IgG-AF546 (1:200; Invitrogen, Carlsbad, CA) in 7% goat serum/PBS for 1 hour at room temperature, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole. All the images were acquired using a Leica DMi8 Thunder Imager fluorescent microscope and analyzed using the Fiji/ImageJ software. For colocalization analysis, a plot profile tool was used to measure and generate the intensity plots of the boxed regions, and Coloc2 plug-in was used to quantify colocalization parameters using Pearson’s correlation efficiency recorded in Fig. 7 and fig. S8.