2.7. Immunofluorescence
After deparaffinating, brain sections were processed for antigen retrieval by heat mediation in a citrate buffer for 20 min. For immunostaining with 4-HNE, slices were blocked with 10% donkey serum for 30 min followed by incubation with the primary antibody against 4-HNE overnight (ab46545, 1:50 dilution, Abcam, UK). Following three washes with PBS, samples were probed with Alexafluor 488 secondary antibody (A21206, 1:200, Thermo Fisher Scientific, United States) for 1 h. Negative control was performed by omitting the primary antibody. Fluorescent signals were captured using AXIOPHOT Zeiss microscope. Data acquisition was carried out by Leica Application Suite 4.9.0 program.