Figure 1. Dual immunofluorescence localization of PSD-95 and pCaMKII T286/287 was used to map colocalization across 42 sample zones in rostral hippocampus. A, Representative images show immunolabeling for pCaMKII alone (left, red), and for pCaMKII plus PSD-95 (right, red and green, respectively) in stratum radiatum (SR) of field CA1b. Scale bar, 2 μm. Note, both antigens are exclusively and densely localized to small puncta, some of which were double labeled (arrows, 2 examples of colocalization). B, Plot shows the immunofluorescence intensity frequency distribution for pCaMKII+ labeling colocalized with PSD-95 (mean values from 3 representative cases). The vertical line marks the intensity value above which labeling was considered intense; elements with this pCaMKII immunolabeling intensity, or higher, were counted as being double labeled in calculations of the percentage PSD-95+ synapses enriched in pCaMKII. C, PSD sizes were distributed according to a Poisson curve (R2 = 0.84). The intensity of pCaMKII labeling colocalized with PSDs did not correlate with PSD size (R2 = 0.0018, p = 0.74). D, Photomicrograph of a rostral hippocampal section processed for the Timm's stain for heavy metals (brown to black) and Nissl staining (violet) to illuminate major lamina and cellular layers, respectively. Sampling zones used for automated counting of pCaMKII+ and PSD-95+ elements are indicated with dotted lines. Each of the major hippocampal subdivisions (CA1, CA3ab, CA3c, DG) were divided into 4–6 zones as illustrated for CA1 SR: these numbered zones (1–5) extended across the three lamina in fields CA1 and CA3. Numbering within CA3 and the DG molecular layer began with CA3a and the lateral aspect of the upper leaf, respectively. LM, Lacunosum-moleculare; SO, stratum oriens; UL, upper leaf of the DG molecular layer; LL, lower leaf of the DG molecular layer.