H3k9ac

Rats and mice were perfused with 0.9% NaCl and Zamboni’s fixative transcardially [60 (link)]. Lumber DRG 4 to 6 and spinal cord dorsal horn were harvested and post-fixed with Zamboni’s fixative for 2 h. Cryoprotection was achieved while using 30% sucrose in phosphate buffered saline (PBS) overnight. The tissues were then sectioned and washed with PBS, incubated with blocking solution (PBS with 1% normal goat serum and 0.3% Triton X-100) for 1 h, and further washed before being incubated with the primary antibody anti-HMGB1 (1:400; Cell Signaling, Danvers, MA, USA), H3K9ac (1:500; Epigentek, Farmingdale, NY, USA), CXCR4 (1:2000; ThermoFisher Scientific, Waltham, MA, USA), NeuN (1:800; Millipore-Sigma, St. Louis, MO, USA) and GFAP (1:1000; Santa Cruz Biotechnogy, Santa Cruz, CA, USA) overnight at 4 °C. Following three washes, Alexa Fluor 594 goat anti-rabbit IgG and 488 goat anti-mouse IgG at a concentration of 1:1000 were used as secondary antibodies (Molecular Probes, Eugene, OR, USA) for one hour incubation, washed three times, and stained with DAPI. The slides were finally mounted with Fluoromount G (Electron Microscopy Sciences, Fort Washington, PA, USA).

ChIP was performed while using EpiQuik Tissue ChIP Kit (Epigentek, Farmingdale, NY, USA) consistent with a modified manufacturer protocol. The dissected ZDF spinal cord samples were excised into smaller pieces (1–2 mm³). The sliced and crushed samples were incubated with fresh 1% paraformaldehyde to cross-link proteins to DNA on a shaker for 15–20 min at 25–30 °C. Subsequently, instructions from the protocol were followed accordingly. Chromatin fragments were generated from the sheared lysates procedure by sonication. The input control for PCR was saved from the sonicated chromatin. The chromatin was then immunoprecipitated for 2 h at RT with antibody against H3K9ac (Epigentek, Farmingdale, NY, USA) or control IgG. The protein—DNA immunocomplexes were precipitated while using spin columns and several steps were followed, as instructed. The purified DNA was isolated with specific immunoprecipitate or with negative control IgG and used as a template for PCR to amplify the HMGB1 promoter sequences. A 197-base pair fragment were amplified while using the ChIP primer sequences: 5′-CTCCAGGAAACGGCTTTGTA-3′ and 3′-TCCACAGAGTTAGTTCCAGAGGA-5′ corresponding to the core HMGB1 promoter.