Anti h3cit

Spleen neutrophils were isolated with Percoll (Solarbio, Beijing, China) gradients as described (23 (link)). NETs assay was performed as protocols published (24 (link)). Neutrophils were resuspended in RPMI-1640 containing 5-10% FBS and plated at 5×10⁵ cells per well in 24-well plates (Thermo Fisher, Massachusetts, US). After incubation for 0.5-1 h, the cells were stimulated with PMA (8 μM, MedChemExpress, New Jersey, USA), ionomycin (10 μM, MedChemExpress, New Jersey, USA) or fecal microbiota (2×10⁷ CFU), and the neutrophils were incubated for an additional 3 h to form NETs. Cells were stained with SYTOX green (1:1000, KeyGEN BioTech, Nanjing, China) for NETs quantification or, in some experiments, fixed in 2% paraformaldehyde, permeabilized, blocked, and stained with anti-H3cit (1:1000, Abcam, cat. no. ab5103) and anti-rabbit secondary antibodies (1:200, Servicebio, Wuhan, China). Images were acquired on an Axiovert 200 M wide-field fluorescence microscope (Nikon) with a coupled camera. The percentages of H3cit high cells and NETs were determined from five or six nonoverlapping fields per well, and the average was taken from 2-3 biological repetitions in every experiment.

For immunofluorescent staining, paraffin slides of endometrial/decidual tissues were processed and incubated with primary antibodies, including anti-CD15 (Abcam, USA; 1:1000) and anti-H3Cit (ab5103, USA; 1:1000), before being stained with fluorescent secondary antibodies and DAPI. The images were acquired with a fluorescence microscope (Olympus, Japan), and the number of endometrial/decidual samples with Nets positive was counted. Meanwhile, the paraffin slides were processed and stained with PAD4 antibody (Affinity, USA; 1:200) before being stained with secondary antibodies and chromogen substrate for immunohistochemical staining.