PF11 distinguishes postsynaptic PSD-95 from extrasynaptic PSD-95. (A and B) Hippocampal neurons were stained triply with hPF11, PSD-95, and vGlut1 antibodies. Asterisks in intensity profiles (B) indicate postsynaptic hPF11 signals (green) colocalized with total PSD-95 (red) and vGlut1 staining (blue), and arrows indicate extrasynaptic PSD-95 signals devoid of hPF11 and vGlut1 signals. A dendritic region indicated by a thick black bar in the intensity profile is magnified (B). Representative profiles from ten repeats (three neurons) are shown (B). (C and D) Hippocampal neurons were treated with or without 2-bromopalmitate (2-BP; 100 µM, 6 h) and stained with hPF11 and PSD-95 antibodies. Representative dendritic regions are shown (C). 300–400 dendritic clusters and 30–40 soma clusters from 5 neurons were analyzed for fluorescence intensities of green and red channels (D). palm, palmitoylated PSD-95. n.s., not significant; ***, P < 0.001, determined by one-way ANOVA with post-hoc Tukey’s test. Bars: (A) 10 µm; (B) 1 µm; (C) 5 µm.