Ab51997

Tadpoles were anesthetized in 0.02% MS-222, and fixed in 4% paraformaldehyde (PFA, pH 7.4) at 4°C overnight or room temperature for 2 h. Tadpoles were rinsed with 0.1 M PB and immersed in 30% sucrose overnight for dehydration. Animals were embedded in optimal cutting temperature (OCT) media and cut into 20 μm cryostat sections with a microtome (Microm, HM550 VP). Sections were rinsed with 0.1 M PB for 2 × 20 min, and permeabilized with 0.3% Triton X-100 in PB, and blocked in 5% goat serum for 1 h before incubating with primary antibodies at 4°C overnight. For primary antibodies, we used the antibodies of SOX2 (1:100, Rabbit, ab97959, Abcam), HDAC2 (1:200, Rabbit, ab137364, Abcam), HDAC3 (1:200, Rabbit, ab16047, Abcam), H2BK5Ac (1:600, Rabbit, ab40886, Abcam), H4K5Ac (1:600, Rabbit, ab51997, Abcam), H4K8Ac (1:600, Rabbit, ab45116, Abcam), H4K12Ac (1:600, Rabbit, ab46983, Abcam), H4K16Ac (1:600, Rabbit, ab109463, Abcam), H3K9Ac (1:600, Rabbit, ab10812, Abcam), and BLBP (1:200, Rabbit ab324223, or Mouse, ab131137, Abcam). Sections were rinsed with PB and incubated with the secondary antibody (FITC or Rhod) for 1 h at room temperature. After sections were counterstained with DAPI, mounted on slides and sealed with clear nail polish, the immunofluorescent images were collected using a confocal microscope (LSM710, Zeiss, Germany).

Cells (2 × 10⁶) in a 100 mm culture dish were treated with 1% formaldehyde to cross-link proteins to DNA. The cell lysates were sonicated to the shear DNA to 300–1000 bp fragments. Equal aliquots of chromatin supernatants were incubated with 1 μg of an anti-MYC (#9402, CST), anti-KAT5 (ab23886, Abcam), anti-acetyl-histone H3 (Lys14) (D4B9) Rabbit mAb (#7627, CST), Anti-histone H4 (acetyl K5) (ab51997, Abcam), anti-histone H4 (acetyl K8) (ab15823, Abcam), anti-acetyl-histone H4 (Lys12) (D2W6O) Rabbit mAb (CST#13944), anti-acetyl-histone H4 (Lys16) (E2B8W) Rabbit mAb (CST#13534), and anti-IgG antibodies (Millipore, Billerica, MA, USA) overnight at 4 °C with rotation. After reversing the cross-linking of protein-DNA complexes to liberate the DNA, the human CDK2 promoter was amplified using real-time PCR. The primer sequences used are listed in Supplementary Table S1.

gADSCs–pDsRed2-1, gMDSCs–pDsRed2-1 and gFFCs–pDsRed2-1 transgenic embryos at the two and four cell stage were studied using 5-methylcytosine (ab124936; Abcam Cambridge, UK), H4K5 (ab51997; Abcam, Cambridge, UK), H4K12 (ab104127; Abcam Cambridge, UK) and H3K18 (ab1191; Abcam Cambridge, UK) with an immunohistochemical method.

Western blots were performed with the following antibodies: DNAJC9 (1:1000, ab150394, Abcam), HA (1:3000-5000, C29F4 #3724, Cell Signaling Technology), DAXX (1:250, HPA008736, Sigma), H3 (1:500-1000, ab10799, Abcam), H4 (1:1000, ab17036, Abcam), Tubulin (1:10000, ab6160, Abcam), H3K9me3 (1:1000, ab194296, Abcam), H3K9me0 (1:1000, 91155, Active Motif), NASP (1:1000, ab181169, Abcam), H4K20me0 (1:500, ab227804, Abcam), H4K20me2 (1:500, C15200205, Diagenode), H4K5ac (1:1000, ab51997, Abcam), Actin (1:5000, A5316, Sigma), SUV39H1 (1:500, 8729, Cell Signaling Technology), SUV39H2 (1:500, ab107225, Abcam), SETDB1 (1:1000, ab107225, Abcam, IP 2 ug, 11231-1-AP, Proteintech), ASF1 (1:1000), ADNP (1:500, Bethyl), SPT2 (1:1000, Abcam), UBR7 (1:1000, Bethyl), CBX3 (1:500, ab56978, Abcam), ERCC6/ERPG3 (1:200 Santa Crutz), FLAG (1:1000, F7425, Sigma).

Equal amounts of cell lysates were loaded on 10% or 13% polyacrylamide gels and transferred to a PVDF membrane. The detection of proteins was performed using primary antibodies against RNF8 (Abcam, ab105362), β-actin (Abcam, ab129348), Ub-H2A (Merck Millipore, ABE569), Ub-H2B (Merck Millipore, MABE453), and H4 (Abcam, ab51997) and HRP-conjugated anti-rabbit or anti-rat secondary antibodies (Abcam, ab6721, ab6728). Densitometry was performed using Photoshop CC.

After NET induction, neutrophils were fixed with 4% paraformaldehyde for 15 min at room temperature, washed three times with PBS, and then permeabilized in 0.1% Triton X-100 for 10 min. Next, the cells were blocked with PBS containing 5% bovine serum albumin (BSA) for 30 min, then incubated with anti-MPO (1:300, AF3667; R&D Systems, USA) and anti-acetylated histone 4 (1:300, ab51997; Abcam, UK) in a blocking buffer overnight at 4 °C. After washing three times with PBS, the cells were again incubated with fluorochrome-conjugated secondary antibodies (1:1000, green fluorescence Alexa Fluor 488 dye plus a red fluorescence Alexa Fluor 568 dye; Invitrogen) for 40 min at room temperature, and then counterstained with ProLong Gold antifade reagent with 4′, 6-diamidino-2-phenylindole (DAPI) (P36931; Invitrogen) before mounting. The cells were observed and photographed using a confocal microscope observer (ZEISS Axio Scan, Z1), and image processing and analysis were performed using the Zen blue edition software.