Double immunofluorescence staining protocol

Double IF staining was performed as described (22 (link)). Briefly, brain tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm. After dewaxing, the sections were permeabilized in Immunostaining Permeable Buffer (Beyotime). After three washes in PBS, the sections were blocked in Immunostaining Blocking Buffer (Beyotime) for 1 h at RT and incubated with primary antibodies at 4°C overnight: rabbit anti-p-PERK (Abcam), rabbit anti-p-eIF2α (Abcam), mouse anti-NeuN (Abcam). After three washes in PBS, the sections were incubated with donkey anti-rabbit IgG Alexa Fluor 488 (Invitrogen) and donkey anti-mouse IgG Alexa Fluor 555 (Invitrogen), for 1 h at RT. Finally, counterstaining was carried out with DAPI and a U-RFL-T fluorescence microscope (OLYMPUS, Japan) was utilized for analysis.