Figure 7.

Detection of ASC and GSDMD in brain cells. (A) ASC immunostaining showed that ASC expression was increased in the hippocampal area of HO₂-EV–injected and LO₂-EV–injected brains (red arrows) compared with the hippocampal areas of control EV–injected brains. Representative red arrows point to focal enlarged areas of ASC⁺ cells in the red boxes. Objective magnification, 20×. Scale bars, 50 μm. (B) Double immunofluorescent staining for ASC (green), AIF1 (allograft inflammatory factor 1; red), and DAPI (blue). ASC was colocalized with AIF1 in the HO₂-EV–injected hippocampus (white arrow and white box). Scale bars, 50 μm. (C) Double immunofluorescent staining for ASC (green), GFAP (glial fibrillary acidic protein; red), and DAPI (blue). ASC was colocalized with GFAP in control EV–injected, LO₂-EV–injected, and HO₂-EV–injected hippocampi (white arrows and white arrows). Objective magnification, 20×. Scale bars, 50 μm. (D) GSDMD immunostaining showed that its expression was increased in the hippocampal area of HO₂-EV–injected brains (red arrows) compared with the hippocampal areas of control EV–injected and LO₂-EV–injected brains. Representative focal enlarged areas of GSDMD⁺ cells are in the red boxes. (E) Double immunofluorescent staining for GSDMD (green), AIF1 (red), and DAPI (blue). GSDMD was colocalized with AIF1 in the hippocampus of all three groups, but only the LO₂ and HO₂ groups had microglial cells exhibiting active forms (white arrows and white boxes). (F) Double immunofluorescent staining for GSDMD (green), GFAP (red), and DAPI (blue). GSDMD was colocalized with GFAP in the hippocampal areas of three groups (white arrows and white boxes). (G) Double immunofluorescent staining for GSDMD (green), SOX10 (red), and DAPI (blue; not shown). GSDMD was colocalized with SOX10 in the hippocampal areas of all three groups (white arrows and white boxes). Objective magnification, 20×. Scale bars, 50 μm.