Vectashield mounting medium with dapi

The NCM460 cells cultured in collagen-coated cover slides were treated with rCPB2 toxin for different times (5 min, 30 min, 2 h, 4 h, 6 h, and 12 h) for evaluating the binding of rCPB2 to cell membranes. The rCPB2-treated cells were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min, washed in PBS for 3 × 5 min, and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. Nonspecific antibody binding was blocked using 5% normal donkey serum in PBS for 1 h at room temperature, after which primary antibodies against CPB2 toxin were applied at a 1 : 100 dilution in PBS and incubated at 4°C overnight. Primary antibody binding was detected using the FITC-labeled donkey-anti-mouse IgG secondary antibody (1 : 500) (Thermo, Rockford, USA). After extensive washing, cell membranes were mounted by Annexin A2 Polyclonal Antibody (1 : 200) (SanYing Biotechnology, China) as primary antibody, and Rhodamine- (TRITC-) conjugated goat anti-rabbit IgG as secondary antibody (1 : 250) (SanYing Biotechnology, China). After extensively washing, the slides were mounted for fluorescence in Vectashield Mounting Medium with DAPI (Thermo, Rockford, USA). Images were acquired using a Leica TCS SP2 A0BS Confocal System and processed on Leica Confocal Software v.2.6.1 (Leica, Germany).

Anterior lobe of lungs were recovered from infected mice, fixed with 3.7% phosphate-buffered formalin, embedded in paraffin, sectioned in 3μm thickness sections and stained with hematoxylin and eosin (H&E). Immunofluorescence was performed on formalin-fixed tissue sections as previously described [24 (link)]. Briefly, antigens were unmasked and blocked with BSA and Fc-Block, and endogenous biotin was neutralized. Sections were probed with purified rabbit anti-NOS2 (M-19, Santa Cruz Biotechnology) followed by a secondary Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen). Vectashield mounting medium with DAPI (Invitrogen) was used to detect nuclei. Images were obtained with an Olympus BX61 microscope and were recorded with a digital camera (DP70).