Rabbit anti hmgb1 monoclonal antibody

Hippocampal tissues were extracted from mice following the indicated treatment protocol (Sect. 4.4), incubated in RIPA lysis buffer (Beyotime, Haimen, China) supplemented with the protease inhibitor PMSF, homogenized by homogenizer and centrifuged at 12000 rpm for 15 min at 4 °C. Subsequently, the supernatant was collected and the protein concentration determined using a BCA protein assay kit (Beyotime). Lysate samples were mixed with 5 × (v/v) loading buffer and boiled in a water bath for denaturation. The denatured protein samples (100 μg per gel lane) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk, incubated with rabbit anti-HMGB1 monoclonal antibody (1:1000, abcam, UK) and rabbit anti-TLR4 polyclonal antibody (1:1000, abcam, UK) overnight at 4 ℃, rinsed with tris-buffered saline plus Tween-20 (TBST), incubated with Dylight 800 goat anti-rabbit IgG at room temperature (r/t) for 1.5 h, and rinsed again in TBST. Immunolabeling was recorded using an Odyssey scanner (LI-COR, USA) and quantified using ImageJ (NIH, Bethesda, MD, USA).

Immunofluorescence assays (IFA) for HMGB1 localization and translocation in ANA1 and mouse PMΦs were performed as described previously (Wang et al., 2014 (link)). Briefly, cells cultured on coverslips and subjected to different treatments were fixed, permeabilized, and blocked prior to incubation with the rabbit anti-HMGB1 monoclonal antibody (ABcam, UK, diluted 1:200 in 1% BSA-PBS) for 1 hr at 37°C. The coverslips were washed and then incubated for 1 h at 37°C with FITC-conjugated goat anti-rabbit IgG (H+L) diluted to 1:50 in PBS with 1% BSA (Proteintech, USA). Nuclei were stained with Hoechst 33258 (Sigma–Aldrich, USA) and coverslips were subsequently mounted onto slides. Fluorescence images were obtained through the Leica microsystem (Leica TCS SP5 II, Germany) and epifluorescence optics were obtained using an oil immersion lens with 63× magnification. Collected images and MIFs (mean index of fluorescence) were processed and analyzed using the LAS AF lite 2.2.0 software (Leica). Mouse anti-TgHMGB1a antibodies were used to trace the T. gondii (Wang et al., 2014 (link)).