HE staining
The lung, liver, and intestine tissues of the mice were fixed with 10% neutral formalin, embedded with paraffin, sliced, stained with hematoxylin-eosin (HE) and observed under a light microscope. A semi-quantitative analysis was carried out in a blinded fashion by an experienced pathologist who was unaware of the groups to quantify the results, defined as: 0 = normal, + = mild, ++ = moderate, +++ = severe histological changes. The tissues and parameters assessed included the lungs (thickening of the septum, edema, congestion and intestinal leukocyte infiltration); the liver (enlarged sinusoids, increased volume of endothelial cells, luminal leukocyte infiltration, hydropic degeneration, Kupffer cell hypertrophy and hyperplasia) and the intestine (edema of mucosal villi, infiltration of necrotic epithelial and inflammatory cells, injury of intestinal glands, blood and lymph vessels expanded).
Immunohistochemistry
CitH3 has been identified as an important biomarker of NETs. Thus, immunohistochemistry analysis was performed to determine the expression levels of citH3. Immunohistochemistry was performed using standard protocols. Briefly, paraffin sections were deparaffinized and rehydrated, washed three times with PBS (PH7.4). Sections were placed in 3% hydrogen peroxide solution was incubated for ten minutes. After washing with PBS, 4% goat serum was added dropwise to block, at room temperature for 30 minutes. Add a sufficient amount of primary antibody (CitH3, Abcam, Cambridge, MA, USA) to the section and place it in a humidified box, and incubate at room temperature for 2 hours. After washing three times with PBS, add secondary antibody (goat anti-rabbit IgG, Abcam, Cambridge, MA, USA) and incubate for 30 minutes. Finally, stain with DAB solution and observe under the microscope, the positive signal is brown.