Gt10312

Mice were perfused with normal saline followed by a fixative reagent containing 4% paraformaldehyde in 0.1 M PBS; brain samples were harvested and protected in sucrose solution (30%) overnight and embedded in optimal cutting temperature compound (Solarbio, China) at −80°C. Then, 4-μm-thick coronal sections from of the brains were made in a cryostat, we sliced the entire brain but just focused on the hippocampus in this experiment. Sections were blocked in 5% BSA for 1.5 hours at room temperature and incubated in primary antibodies at 4 °C overnight. For double immunofluorescent staining, rabbit anti-NeuN (1:200; ab177487; Abcam) and mouse anti-NeuN (1:200; Millipore; MAB377) for neurons, rabbit anti-IBA1 (1:50; 10904-1-AP; Proteintech) and mouse anti-IBA1 (1:200; GT10312; GeneTex) for microglia, mouse anti-GFAP (1:200; SC-33637; Santa Cruz Biotechnology) and rabbit anti-GFAP (1:200; ab33922; Abcam) for astrocytes were used as specific cellular markers. The sections were then incubated with Alexa Fluor 488 (1:200) and Alexa Fluor 594 (1:200; Invitrogen) for 1.5 hours at 37 °C. The staining was examined with a fluorescence microscope (OLYMPUS, BX53 MicroPublisherTM 5.0 RTV, Japan).

Mice were anesthetized 48 h after reperfusion, transcardially perfused with phosphate-buffered saline (PBS), and fixed with 4% paraformaldehyde. Their brains were rapidly removed and postfixed with 4% paraformaldehyde. Brains were then immersed in 20% and 30% sucrose overnight at 4°C, embedded in Tissue-Tek OCT compound (Sakura, Japan), and subjected to section on a freezing microtome. The sections (10 μm) were washed with PBS and blocked in QuickBlock™ immunostaining blocking solution (Beyotime, China) for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: rabbit anti-Sertad1 (1:200, ARP34309_T100; AVIVA, USA), mouse anti-NeuN (1:100, 66836-1-Ig; Proteintech, China), mouse anti-GFAP (1:200, ab279290; Abcam, USA), mouse anti-Iba1 (1:500, GT10312; GeneTex, USA). After washing, the sections were incubated with Alexa Fluor Plus 488 goat anti-rabbit secondary antibody (1:200, A32731; Thermo Fisher Scientific, USA) or Alexa Fluor Plus 555 goat anti-mouse secondary antibody (1:200, A32727; Thermo Fisher Scientific) at 37°C for 1 h. DAPI (Beyotime) was used to stain cellular nuclei at 37°C for 10 min. Finally, the sections were photographed using a microscope with a digital camera (Olympus, Japan).

Lipopolysaccharide (LPS, L2630) and sodium butyrate (NaB, B5887) were purchased from Sigma. ML385 was purchased from MCE. PTX was purchased from GlpBio. PGE₂ ELISA kit was purchased from Shanghai Ruifan Biological Technology Co. Ltd. Antibodies against i-NOS (ab178945), COX-2 (ab179800), BDNF (ab108319), HO-1 (ab52947), NQO1 (ab80588), Keap-1 (ab227828) and GAPDH (ab181602) were purchased from Abcam. Antibodies against Nrf2 (GTX103322) and Iba-1 (GT10312) were purchased from Genetex. Antibodies against GPR109A (sc-377292) was purchased from Santa cruz.