Ncnp24

The mice brain sections were separated and then fixed by 4% paraformaldehyde (Sigma-Aldrich, St Louis, Missouri, MO, USA). After three-times washing with PBS, the tissues were then permeabilized with the blocking buffer (0.3% Triton X-100, 10% goat serum in PBS). Then the tissues were incubated with antibodies against Iba-1 (#NCNP24, Wako, Osaka, Japan) or GFAP (#ab4674, Abcam, Cambridge, Massachusetts, MA, USA) overnight at 4 °C and further incubated with relative secondary antibodies for another 1 h, at room temperature. Thereafter, the brain slices were stained with 5 μg/mL of DAPI (Sigma-Aldrich, USA) for 15 min. Images were photographed by using a Leica TCS SP8 confocal laser scanning microscope (Solms, Germany).

For in vivo experiment, 4 h after intraperitoneal injection with lipopolysaccharide and propofol, mice were sacrificed under deep anaesthesia and transcardially perfused with 0.9% saline solution, followed by 4% PFA in 0.1 M PBS. The brains were removed and immersed in 4% PFA for 24 h, and then cryoprotected in 30% sucrose solution in PBS (pH 7.4). Subsequently, serial coronal sections were then prepared using a microtome. For in vitro study, BV2 microglia cells were fixed with 4% Paraformaldehyde (PFA) for 15 min at 37°C. After fixation, slides were washed in PBS, blocked with 5% Bovine albumin and 0.5% Triton X-100 in PBS, and then incubated with primary antibodies (mouse anti-Iba-1: 1:200, NCNP24, Wako, Japan; mouse anti-NeuN: 1:200, ab104224; rabbit anti mouse PFKFB3: 1:200, ab181861; rabbit anti mouse PSD-95: 1:200; ab18258, Abcam, United States) at 4°C overnight followed by secondary antibodies (Alexa Fluor 594-labeled goat anti-mouse IgG: 1:500, ab150116 or Alexa Fluor 488-labeled goat anti-rabbit IgG: 1:500, ab150077; Abcam, United States) as appropriate. Counterstaining was then performed with DAPI (1 ug/mL). The slides were examined under a Confocal Laser Scanning Microscope (Leica, Solms, Germany).

Brain and lung hematoxylin and eosin staining and immunofluorescence were performed on 5-mm-thick, 4% paraformaldehyde-fixed, paraffin-embedded slices. Antibodies against mouse Ym1 (60130, StemCell Technologies), CD11b (MAB1124, R&D Systems), and Iba1 (NCNP24, Wako) were used as primary antibodies, with the corresponding secondary antibodies labeled with FITC or Alexa Fluor® 594. In addition, nuclei of cells were displayed by DAPI staining. All tissue slices were examined with laser-scanning confocal microscopy (Zeiss LSM780; Germany).