“…For immunofluorescence staining, sections were rinsed three times in 0.1 M PBS for 10 min, followed by a 1 h incubation in 0.1 M PBS, 5% v/v normal donkey serum (NDS), 5% v/v normal goat serum (NGS), and 0.5% v/v Triton-X for blocking and permeabilization. Primary antibodies against NeuN, GFAP, CD31, GLUT-1 and pS129 (EP1536Y, 81A and MJR-R13 clones), were incubated overnight at 4°C (in 0.1 M PBS, 3% v/v NDS, 3% v/v NGS and 0.3% v/v Triton-X) (Kecheliev et al 2023; Sobek et al 2023). The sections were then rinsed three times for 10 min each in 0.1 M PBS before being incubated for 2 h in species specific secondary antibodies (suspended in 0.1 M PBS with 3% v/v NDS, % v/v NGS and 0.3% v/v Triton-X).…”