4.7. Immunohistochemistry

Brain sections were permeabilized with 0.3% Triton X-100 (T8787, Sigma, St. Louis, MO, USA) for 10 min, blocked with 3% bovine serum albumin (BSA, A3294, Sigma, St. Louis, MO, USA) for 1 h at room temperature, and then incubated overnight at 4 °C with a primary antibody anti-HMGB1 in combination with cell-specific markers anti-NeuN for neurons, anti-GFAP for astrocytes, or anti-CD11b for microglia (Table 1). After washing, sections were incubated with the corresponding secondary antibodies (Table 1) for 1 h at room temperature. Next, sections were mounted with Vectashield antifade mounting medium with DAPI (H-1200, Vector Laboratories, Burlingame, CA, USA). Fluorescence images of the whole dorsal hippocampus were acquired using the VS120 virtual microscopy slide scanning system (Olympus).

Antibodies used for immunofluorescence/Western blotting.