Alexa fluor 488 goat anti rabbit igg

Following the completion of behavioral experiments, including righting reflex and EEG monitoring, mice were deeply anesthetized using sevoflurane and subsequently perfused with 4% paraformaldehyde (PFA), followed by 0.9% saline. The brain slices were washed three times (5 min each) with 1× PBS to remove OCT. Subsequently, they were incubated with BSA at room temperature for 30 min. After that, the diluted primary antibodies, including glutamate antibody (1:1000, Sigma, G6642, rabbit) and Anti‐c‐fos antibody (1:1000, Abcam, ab208942, mouse), were applied and incubated overnight at 4°C. The slices underwent three 5‐min washes with 1× PBS to remove the primary antibodies. Secondary antibodies (1:500, labeled with Alexa Fluor 488 goat anti‐mouse IgG, Alexa Fluor 488 goat anti‐rabbit IgG, Cy3‐labeled donkey anti‐mouse IgG, Cy3‐labeled donkey anti‐rabbit IgG from Shanghai Beyotime Biotechnology Co., Ltd.) were added and incubated in the dark at room temperature for 2 h. After another wash with 1× PBS, the slices were covered with DAPI and incubated for 15 min. Finally, the images were captured using a fluorescence microscope (Revolution, Echo, America). Experimenters conducting the microscopic analysis were blinded to the group assignments.

Bone marrow CD115⁺ monocytes (1 × 10⁶/mL) were seeded in 6-well plates and cultured overnight. Based on experimental aims, monocytes were treated with different reagents for about 24 h. Cells were then washed twice with PBS and fixed in 4% paraformaldehyde for 10 min, then washed again with PBS. Next, cells were incubated for 1 h at room temperature in buffered normal goat serum to prevent nonspecific binding of antibodies. Next, they were incubated separately overnight with an antibody purchased from CST against Ki67 (dilution 1 : 400; Boston, USA, catalog number 9129T), followed by incubation with Alexa Fluor 488 goat anti-rabbit IgG (dilution 1 : 500; Beyotime, Jiangsu, China, catalog number A0423) for 1 h at 37°C. Thereafter, cells were washed with PBS. 40,6-Diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei (blue) at a concentration of 1.43 μM (catalog number D8417; Sigma). Photomicrographs were taken with a DMI3000B camera (Leica, Germany).

BV2 cells were fixed in 4% paraformaldehyde for 10 min, infiltrated with 0.5% Triton X-100 (Bio-Rad) for 20 min after washing 3 times with PBS and then incubated with anti-p65 antibody (1:400) at 4°C overnight, followed by Alexa Fluor 488 goat anti-rabbit IgG (1:1000) for 1 h and DAPI (1:1000, Beyotime, China) for 20 min. Images were acquired with a confocal microscope (FV-OSR, Olympus, Japan) and quantitated using ImageJ.

Brain cryosections and astrocytes coverslips were immunostained with following primary antibodies: rabbit anti-YAP polyclonal antibody (1:100, Santa Cruz Biotechnology, USA), rabbit anti-Ki67 polyclonal antibody (1:500, Abcam), rabbit anti-GFAP polyclonal antibody (1:1000, Servicebio, China), mouse anti-VIM monoclonal antibody (1:500, Servicebio), mouse anti-GFAP monoclonal antibody (1:500, Servicebio). Secondary antibodies used included Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 555 donkey anti-rabbit IgG, Alexa Fluor 555 donkey anti-mouse IgG and, and Alexa Flour 647 goat anti-mouse IgG (1:500, Beyotime). Nuclei were stained with DAPI (1:3000, Beyotime). The fluorescence images were observed and analyzed by confocal laser-scanning microscope (Leica).