Ab131260

After treatment with 4% paraformaldehyde for 20 min, NETs were incubated with anti-myeloperoxidase (MPO) antibody [EPR20257] (ab208670, Abcam, Cambridge, MA), and anti-neutrophil elastase (NE) antibody [EPR7479] (ab131260, Abcam), respectively. When the nucleus was stained, the NETs were observed under the Olympus IX81 inverted fluorescence microscope and the images were collected. Three different visual fields were randomly selected under the same conditions, and the total cells number in a single visual field was calculated by Image J 1.8.0 software (National Institutes of Health, USA). The NETs productivity was calculated according to the ratio of the NETs number in visual field to the total cells number.

Serially cut frozen slides were air-dried and fixed in prechilled 100% acetone). Slides were rinsed in distilled water and then washed in TBS-T. Endogenous peroxidase activity was quenched via incubation with 5% (v/v) H₂O₂ (Sigma-Aldrich, H3410) in dH₂O. After 30 minutes, the slides were washed and incubated in serum-free protein block (DAKO, X0909) for 30 minutes, after which excess liquid was removed. Serial sections were then incubated with 1:1000 (v/v) anti-MPO (mouse, monoclonal; Abcam, ab25989) or 1:1000 (v/v) anti-NE (rabbit, monoclonal; Abcam, ab131260) or 1:500 (v/v) anti-CitH3 (rabbit, monoclonal; Abcam, ab219407) for 1 hour in antibody diluent (bovine serum albumin [1% w/v], Sigma-Aldrich, A7906) and Triton-X100 (0.5% v/v, Sigma-Aldrich, 270733) in TBS-T. Slides were further washed in TBS-T and incubated with horse radish peroxidase (HRP)-coupled secondary antibody (anti-mouse, DAKO, K4001; anti-rabbit, K4003) for 30 minutes. Following washing in TBS-T, slides were incubated for 10 minutes with Opal690 fluorophore (PerkinElmer, FP1497) in tyramide signal amplification (TSA) buffer (PerkinElmer, FP1498, 1:50 v/v dilution in 1x plus amplification buffer) and subsequently washed. Specimens were incubated in a 1:800 (v/v) dilution of Spectral DAPI for 10 minutes, washed, and then cover-slipped with fluorescence mounting medium.

We utilized a previously established ELISA assay for the detection of elastase-DNA complexes^{41 (link)} with slight modifications. A rabbit polyclonal anti-neutrophil elastase antibody (1:500; ab131260, Abcam, MA, USA) in 100 mM of bicarbonate/carbonate coating buffer (50 μL in total) was coated onto 96-well microtiter plates (ThermoFisher Scientific, MA, USA) overnight at 4 °C. After blocking in 1% BSA for 1 h, the mixture of 50 μL of human plasma and 50 μL of PBS containing 0.3% BSA, 0.05% Tween 20, and 5 mM EDTA was loaded per well and incubated at room temperature for 1 h. After washing three times with PBS containing 0.05% Tween 20 and 5 mM EDTA (PBST-EDTA), a horseradish peroxidase conjugated anti-DNA antibody (1:100; D5425–3–100, Zymo Research Corporation, CA, USA) in PBST-EDTA (100 μL in total) was added to each well and incubated at room temperature for 1 h. After washing three times with PBST-EDTA, the peroxidase substrate (Glo Substrate Reagent Pack (DY993), Bio-Techne Corporation/R&D Systems, MN, USA) was added. The intensity of chemiluminescence was measured using a plate reader (TECAN Infinite M200Pro, Tecan Group Ltd., Switzerland). For analysis, the luminescence of the blank well was subtracted from the luminescence of each sample.

LA was embedded in paraffin and used for immunohistochemistry or immunofluorescence (IF) analyses. Masson’s trichrome was used to compare the collagen volume. For IF staining of heart sections, primary antibodies anti-MPO (ab90810 or ab134132, Abcam, UK), neutrophil elastase (NE, ab131260, Abcam, UK) and cit-H3 (ab5103, Abcam) antibodies were used to identify NETs in LA. Cells were fixed with 4% paraformaldehyde for 15 min and NETs were fixed for 30 min to 1 h. Followed by permeabilization with 0.5% Triton-X-100 (BioFroxx, Germany) for 10 min, and sealing with the corresponding serum (Solarbio, China). Besides using primary antibodies to identify NETs, anti-LC3B (2775 S, CST, USA) and anti-p62 (ab56416, Abcam) antibodies were incorporated for the analysis of autophagy. The primary antibodies were incubated overnight at 4 °C, while the secondary antibodies were incubated at room temperature for 1 h in a dark room. DAPI (10 μg/mL) was used to stain nuclei.