Donkey anti rabbit igg h l alexafluor 488

The mice were anesthetized with 1% pentobarbital sodium and perfused with saline for a few minutes. Then, the mice were perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Brains were dissected and soaked overnight at 4°C in 4% PFA, and then soaked in 30% sucrose at 4°C for 48 h. Brain slices (25 μm) were harvested and incubated with primary and secondary antibodies, and then washed in PBST and flat-mounted. The primary antibodies included anti-NeuN (Millipore, MA, United States), anti-G3BP (Abcam, Cambridge, United Kingdom), anti-SYK (Abcam, Cambridge, United Kingdom), and anti-p-SYK (Abcepta, Suzhou, China). The secondary antibodies included donkey anti-rabbit IgG H&L (Alexa Fluor 488, Jackson Immuno Research, PA, United States), donkey anti-rabbit IgG H&L (Alexa Fluor 594, Thermo Fisher Scientific, MA, United States), and donkey anti-guinea pig (Alexa Fluor 647, Abcam, Cambridge, United Kingdom) antibodies. Primary antibodies were diluted at 1:100 for usage, and the secondary antibodies were applied at a dilution of 1:200. In each group, three hippocampus flat mounts were performed and observed under a confocal microscope (LEICA TCS SP8 X, Germany).