Fluoro gel 2 containing dapi

Pregnant dams from PARP1 heterozygous crosses were injected intraperitoneally with EdU (50 mg/kg) at E13.5. Littermate P0 pups were transcardially perfused at birth with PBS followed by 4% PFA, then whole brains were dissected and postfixed in 4% PFA for 24 h. Brains were then either cryoprotected in 30% sucrose, embedded in OCT, and cryosectioned at a thickness of 20 μm or embedded in 4% agarose and sectioned coronally on a vibratome at a thickness of 50 μm. EdU was visualized using the Click-iT EdU Cell Proliferation Kit (Invitrogen), following the manufacturer’s protocol. Brain sections were subsequently co-stained for the layer VI marker TBR1 (1:250) following the immunofluorescence staining protocol described above. Slides were coverslipped with Fluoro-Gel II containing DAPI (Electron Microscopy Services). Confocal z-stack images through the full depth each section were taken at 20× magnification with a Leica SP8 confocal microscope. Each image was separated into the area below layer VI, the area within layer VI, and the area above layer VI, and the number of EdU positive cells within each defined area was quantified using Fiji by an individual blinded to the genotype. For each biological replicate, three sections and three images per section were quantified and averaged.

Embryonic (2 dpf) and larval (3 dpf) zebrafish were anesthetized in MS‐222 Tricaine Methanesulfonate and head and trunk dissected. Trunk tissue was placed in 20 μL 50 mM NaOH for genotyping. Heads were fixed in 4% paraformaldehyde overnight at 4°C, incubated in 30% sucrose overnight at 4°C, then processed and embedded in Tissue‐Tek OCT (Fisher 4583). Tissues were sectioned at 14–16 μm on a Microm HM 550 cryostat. For BrdU labeling experiments, 2 dpf embryos were incubated in 5 μM BrdU (Sigma B5002) in embryo media⁴⁵ for 2.5 hours, placed in fresh fish water, then sacrificed immediately or at 3 dpf and 5 dpf. To aid BrdU antigen, retrieval tissues were pretreated with 2 M HCl. Antibodies used in this study: rabbit polyclonal anti‐phospho‐Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti‐phospho‐Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05‐806); mouse monoclonal anti‐HuC/D 1:500 (Invitrogen A‐21271); rabbit polyclonal anti‐activated Caspase‐3 1:500 (BD Biosciences 559 565); mouse monoclonal anti‐BrdU 1∶500 (Bio‐Rad MCA2483); Alexa‐594 (Invitrogen A‐11005) and Alexa‐488 (Invitrogen A‐11008) conjugated secondary antibodies 1:500. Tissues were counterstained with 5 μg/mL DAPI, mounted in Fluoro‐Gel II containing DAPI (Electron Microscopy Sciences 17985‐50) and imaged on a Zeiss LSM700 laser scanning confocal.

BMM were plated at 5 × 10⁴ cells per well on a 96-well plate and maintained for 24 h in culture media containing a 1:10 dilution of CMG. Cells were primed with 100 ng/ml LPS for 3 hours and incubated with 15 µM nigericin (positive control) for 30 minutes or BP (1.25–5 mg/ml) or HA crystals (0.25–1 mM) for 1 hour. Cells were incubated with the FLICA^TM FAM-YVAD-FMK probe as recommended by the supplier (30 minutes at 37 °C, 2 washes with PBS/FBS and fixation with 10% buffered formalin. Cells were then counterstained with Fluoro-gel II containing DAPI (Electron Microscopy Sciences). Images were taken with a DP70 Olympus Digital Camera coupled to an IX51Olympus inverted microscope, captured with QCapture Pro software and analyzed with ImageJ software.