2.8. Immunofluorescence assay

The FOXO3a nuclear translocation in vitro was observed via immunofluorescence assay. After OGD/R stimulation and SalA treatment, cells for fluorescence imaging were fixed using 4% paraformaldehyde. And then they were permeabilized using 0.3% Triton X-100. After blocked by 5% BSA, the primary antibody against FOXO3a was added and incubated at 4 °C overnight. Then Alexa Fluor-488 (1:500)-conjugated secondary antibody was added and incubated for 1 h. Finally, Hoechst 33258 was added and incubated for 30 min. The fluorescent images for the observation of nuclear translocation were taken under a microscope.

The brain tissue for frozen sections was placed in a 4% paraformaldehyde solution and fixed. After that, frozen sections were prepared into 5 μm thickness. For TUNEL and NeuN or GLUT1 double-labeled staining. Proteinase K was firstly incubated. Then the buffer solution, reaction solution, saline sodium citrate, 0.5% Triton X-100 and 10% goat serum were added following steps and incubated as planned. Then cells were treated with primary antibody against NeuN or GLUT1 overnight, and the secondary antibody for 1 h. The fluorescent images of TUNEL were taken under a microscope.

For FOXO3a and NeuN staining, brain slices were firstly treated with 0.5% Triton X-100, and then treated with 10% goat serum. The primary antibodies against FOXO3a and NeuN were then used and reacted overnight. Then the secondary antibodies were used and reacted. Finally, after DAPI treatment, the fluorescent images of FOXO3a and NeuN staining were taken under a microscope.