Axioobserver epifluorescence microscope

Three 20× images per section were captured by a blinded researcher using a Zeiss Axio Observer epi fluorescence microscope (locations shown in Supplementary Fig. 2). Iba-1 and GFAP images were captured on the Alexa Fluor 488 and Cy3 channel, respectively.

Mouse embryos were immunostained using an adapted protocol from ref. 31 (link) in 96-well plates. Briefly, embryos were subjected to thinning of the zona pellucida using acidic DPBS (pH 2.5), and fixation in 2% paraformaldehyde for 30 min. Embryos were permeabilized in 0.3% BSA, 0.1% Triton X-100, 0.02% NaN₃ PBS solution. Blocking was carried out in 0.3% BSA, 0.01% Tween-20, and 0.02% NaN₃ in PBS. Embryos were incubated in blocking solution with 1:200 H3K4me3 antibody (Merck Millipore, 04–745) for 60 min at room temperature. After further blocking, embryos were finally incubated with goat anti-rabbit Alex Fluor 488 (Invitrogen, A-11008) or Alexa Fluor 568 for morpholino injected embryos (Invitrogen, A-21069) at 1:200 dilution and placed on a slide in SlowFade Gold with DAPI (Invitrogen). Quantitative measurements of H3K4me3 were obtained using a Zeiss Axio Observer epi-fluorescence microscope with a Coolsnap HQ2 camera. Confocal images were obtained with a Zeiss Axio Observer LSM 710 confocal microscope. Images were processed and quantified in Axiovision and ImageJ software.

The NHC cell line (1×10⁵ cells) was seeded on the underside of 24-well plates for 18 hours and incubated with LPS (5 μg/mL) for 6 hours at 37°C. The supernatant was removed and rinsed with PBS then maintained in NHC medium. Meanwhile, human neutrophils were isolated and preincubated with CellTracker Orange CMTMR Dye (5 μM) for 30 min at 37°C. Transwell inserts with 3.0 μm pore-size transparent PET membranes (FALCON-353096) were placed in a 24-well plate. A C-X-C motif chemokine ligand-8 (CXCL8) antagonist (1 μg/mL) was added to the bottom compartment, and neutrophils (5×10⁵ cells) in NHC medium were added to the top of each Transwell. As a positive control, recombinant CXCL8 (1 μg/mL) was added to the bottom side. All neutrophils were allowed to migrate for 1 hour. The migration of neutrophils into the bottom compartment was monitored using a Zeiss Axio Observer epifluorescence microscope with a 20x objective, and the number of migrating neutrophils was quantified using ImageJ.