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.