Lsm 710 meta inverted laser scanning confocal microscope

Ferret colon tissues were stained for the presence of NETs as previously described (Brinkmann et al., 2016 (link); Doster et al., 2018 (link)). Tissues were deparaffinized and incubated in R universal Epitope Recovery Buffer (Electron Microscopy Sciences) at 50°C for 90 minutes. Samples were then rinsed in deionized water three times followed by washing with TRIS-buffered saline (TBS, pH 7.4). Samples were permeabilized for 5 minutes with 0.5% Triton X100 in TBS at room temperature followed by 3 washes with TBS. Samples were then blocked with TBS with 10% BSA for 30 minutes prior to incubation with 1:50 dilutions of rabbit poly-clonal anti-neutrophil elastase antibodies (MilliporeSigma, Cat. 481001). The following day, samples were washed in TBS followed by repeat blocking with blocking buffer for 30 minutes at room temperature before incubation with 1/00 dilution of Alexa Fluor® 488 conjugated donkey anti-rabbit IgG (Invitrogen) for 4 hours at room temperature. Samples were then washed and incubated with 5 μM Hoechst 33342 for 30 minutes to stain DNA/nuclei. After final washes, slides were dried and cover slipped. Tissues were visualized with a Zeiss LSM 710 META Inverted Laser Scanning Confocal Microscope. Images presented are composites of z-stacked images.