Immunofluorescence detection of NETs

To identify NETs from lung tissues, sections from M. tuberculosis-infected mice or from TB patients were de-waxed and re-hydrated before staining. Sections were treated with Dako Target Retrieval Solution, pH 9 Ready-to-Use (Agilent) at 97 ^oC for 45 min for antigen retrieval, and permeabilized in PBS 0.5% triton x-100. Samples were incubated with a blocking buffer (PBS with 2% BSA and 2% of donkey serum (Sigma-Aldrich)) for 1 h at room temperature (RT) and stained with rabbit anti-mouse antibodies directed against citrullinated histone H3 (ab5103 from Abcam; 1:500) and goat anti-mouse antibodies directed against MPO (AF3667, R&D systems; 1:40) overnight at 4 ^oC. After washing samples with PBS, sections were incubated with secondary donkey anti-rabbit IgG antibodies conjugated with Alexa Fluor 568 (A10042, Invitrogen; 1:200), donkey anti-goat IgG antibodies conjugated with Alexa Fluor 488 (A11045, Invitrogen; 1:200) and DAPI (Invitrogen; 1:1000) for 2 h in the dark at RT. Glass coverslips were mounted onto the slides using Prolong Gold (Invitrogen). Controls were stained with secondary antibody only, and nonspecific fluorescent staining was not detected when secondary antibodies were tested alone. Slides were analysed using Olympus VS120-L100 Slide Scanner and the OLYMPUS OlyVIA software (v2.9). Representative images for each group were exported using QuPath software (v0.2.0-m5)⁷⁵.

The percentage of NET area normalized to MPO signal was calculated by exporting three regions of interest of 2000 × 2000 μm representative of each lung sections from Qupath (version 0.2.0) and analysed with FIJI (v 2.0.0). The images were split into the different channels; the DAPI channel was subjected to the threshold Triangle, a mask was generated to account for the total amount of tissue present in the region of interest. Default and Triangle thresholding was used with the MPO and the CitH3 channel respectively. The percentage of the area was calculated for the different channels based on the generated masks. The measured percentage of CitH3 signal was normalized to the measured percentage of MPO signal and the % CitH3/MPO values obtained from the three different regions were averaged and plotted. Statistical analysis of the plots was obtained by performing a nonparametric Mann–Whitney test.