Anti g3bp

Cells were fixed with 4% paraformaldehyde (4% PFA—Polysciences Inc., Hirschberg and der Bergstrasse, Germany; catalog number: 2878-55-4) in Gibco^TM Dulbecco’s Phosphate-Buffered Saline (DPBS—Thermo Fisher Scientific (Brussels, Belgium), catalog number: 14190250) for 15 min and rinsed three times with DPBS. In total, 5 % normal donkey serum (NDS, Sigma, Machelen, Belgium; catalog number: D9663) in 0.1% Triton X-100 in DPBS was used for blocking at room temperature for 1 h. Primary antibodies were diluted in 2% NDS in 0.1% DPBS Triton and incubated overnight at 4 °C. The following primary antibodies were used: anti-G3BP (1/250, catalog number: ab56574), anti-Yb1 (1/500, catalog number: ab76149) and anti-importin-β1 (1/1000, catalog number: ab2811) all provided by Abcam (Cambridge, UK). The cells were subsequently washed with DPBS and incubated with appropriate secondary antibodies (1:2500; Thermo Fisher Scientific, Brussels, Belgium). Nuclei were stained with Hoechst (NucBlue Live ReadyProbes^TM Reagent Hoechst 33342, Thermo Fisher Scientific, Brussels, Belgium; catalog number: R37605) and images were taken under SP8 confocal microscope (64x; Leica, Wetzlar, Germany; model: SP8 MDi8) excitation lines at 405, 488, 555 and 647 nm.

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).