R universal epitope recovery buffer

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.

Formalin-fixed, paraffin-embedded colonic and ileac tissues were subjected to immunohistochemistry. After deparaffinization and rehydration, antigens were exposed through heat-induced epitope retrieval for 15 min in the R-Universal epitope recovery buffer (Electron Microscopy Sciences, Fort Washington, PA). The slides were blocked for 60 minutes in Hanks’ buffered salt solution containing 1% bovine serum albumin, followed by overnight incubation at 4°C with primary antibodies. Washed samples were incubated with Alexa Fluor (488 or 555)-conjugated secondary antibodies for 60 minutes at room temperature, rinsed with blocking buffer, and mounted on slides with ProLong antifade mounting reagent (Life Technologies). Images were obtained on Zeiss LSM 700 Laser Scanning Confocal Microscope (Carl Zeiss Microscopy LCC, Peabody, MA).