A1 mp storm

HeLa cells were seeded in 35 mm glass-bottom dishes and transfected with an empty vector, HSV-2 ICP27-HA expression plasmid or an influenza virus NS1 expression plasmid. At 24 h post-transfection, HeLa cells were stimulated with or without 100 HAU ml⁻¹ SeV for 16 h. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100. After three washes with PBS, cells were blocked with PBS containing 5% BSA for 1 h at room temperature, and then incubated with rabbit anti-human IRF3 polyclonal Ab and mouse anti HA-tag mAb at a dilution of 1:100 for 1 h at room temperature. After three washes with PBS, cells were incubated with Alexa Fluor 488-labeled Goat Anti-Mouse IgG (H+L) and Alexa Fluor 647-labeled Goat Anti-Rabbit IgG (H+L) at a dilution of 1:50 for 1 h at room temperature. Cells were subsequently washed and incubated with DAPI solution for 10 min at room temperature. Following the addition of 1 ml PBS into the dishes, cells were observed under a Multiphoton Confocal Microscope (Nikon, A1 MP STORM).

Lung samples were fixed with 4% paraformaldehyde, paraffin embedded, and cut into 4-μm sections. Fixed tissue samples were used for hematoxylin and eosin (H&E) staining and indirect immunofluorescence assays (IFA) for the detection of the NETs. For routine histology, tissue sections were stained with H&E. For IFA, slides were deparaffinized and rehydrated, followed by 15 min of heat-induced antigen retrieval with EDTA, pH 8.0, in a microwave oven. The slides were washed with phosphate-buffered saline (PBS)–0.02% Triton X-100 and then blocked with 5% bovine serum albumin at room temperature for 1 h. For NET detection, the multiplexed staining followed the instructions of TSA-Rab kit (0082100100; Panovue). Other antibodies and reagents used were anti-myeloperoxidase antibody (ab9535; Abcam), anti-histone H3 antibody (ab5103; Abcam), and DAPI (10236276001; Roche). The image information was collected using a Pannoramic MIDI system (3DHISTECH) and confocal microscopy (Nikon A1 MP Storm).