Analysis of synaptic proteins via sequential immunofluorescence in hippocampus and dentate gyrus. Data from sequential immunofluorescence from hippocampus (H) and dentate gyrus (DG) at different doses are shown (A and B). The columns represent the fold-changes with standard errors of the mean (SEM) from three biological replicates. The visualisation shows the representative intensity from three biological replicates of 0 Gy and 1.0 Gy regarding MAP-2 (red - Microtubule-associated protein 2), PSD-95 (green - Disks large homolog 4 (DLG4), Hoechst (blue) and merged intensities within the hippocampal region. The MAP-2 / PSD-95 intensity was normalised against nuclear Hoechst intensity in the region of interest. *p < 0.05; **p < 0.01; ***p < 0.001 (unpaired Student’s t-test); magnification: 4x. Representative images of sequential immunofluorescence from hippocampus are shown in panel C. Images indicate the specific binding of secondary antibodies (“negative control”), binding sites of primary MAP-2 antibody saturated via Cy3-Fab-fragment IgG (“MAP-2 + sec. Ab’s”) and sequential immunofluorescence with a single protein detection (“Only MAP-2” and “Only PSD-95”). “Negative control” – only secondary antibodies and Hoechst; “only MAP-2” – primary antibody against MAP-2, Cy3-Fab-fragment IgG secondary antibody, Hoechst; “only PSD-95” – primary antibody against PSD-95, Alexa-fluor IgG secondary antibody, Hoechst; “MAP-2 + sec. Ab’s” – primary antibody against MAP-2, Cy3-Fab-fragment IgG secondary antibody, Alexa-fluor IgG secondary antibody, Hoechst; magnification: 4x.