Mouse anti influenza a nucleoprotein antibody

HMVECs were grown to confluency on collagen I-coated culture slides (AGC TECHNO GLASS Co., Shizuoka, Japan). After the indicated treatment time, cells were washed with phosphate-buffered saline (PBS) and fixed with 4.0% paraformaldehyde for 15 min. After permeabilization with 0.2% Triton X-100 in PBS for 10 min, the cells were washed and probed with the primary antibodies (mouse anti-influenza A nucleoprotein antibody from Abcam, Cambridge, MA, USA; rabbit anti-HMGB1 antibody from Abcam; and rabbit anti-vascular endothelial (VE) cadherin antibody from Abcam) at a 1:200 dilution for 60 min. The coverslips were washed and then incubated with Alexa Fluor 488-conjugated goat anti-rabbit or anti-mouse secondary antibody (1:500 dilution) (Invitrogen Corp., Carlsbad, CA, USA) for 1 h at 20 °C. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) in a mounting medium (Vectashield; Vector Laboratories Inc., Burlingame, CA, USA). Samples were evaluated under a fluorescence microscope (BZ-9000 generation II; Keyence Co., Osaka, Japan). When measuring the cell morphology, each long axis and short axis of 100 cells/field at 40 × magnification were measured on the VE cadherin-stained images using the BZ image analysis software application. The same measurements were performed on four randomly selected fields of view.