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. Author manuscript; available in PMC: 2013 Jun 15.
Published in final edited form as: J Neurosci Methods. 2012 Apr 10;207(2):137–147. doi: 10.1016/j.jneumeth.2012.04.003

Figure 1. Dendrite sampling and flow chart of method used for quantitative analysis.

Figure 1

(A) Discrete dendrites 20μm in length were sampled 50μm away from the cell body of neurons quadruple-stained for NR1, GluR2, Drebrin and MAP2. (B) Flow chart and images showing method used for quantitative analyses by ImageJ NIH software. Images are thresholded for the purpose of illustration. 20μm dendritic segments were sampled (1). Region of interest (ROI) was drawn around spines and the parent dendrite to be analyzed. Signals outside of drawn ROI were erased (2). With background set to luminance of diffuse fluorescence signal, clusters were automatically point-selected and counted (3). Point-selections were converted into individual circular ROIs and centered on each cluster. Mean fluorescence within individual circular ROI was used as a measure of individual cluster fluorescence intensity (4). For colocalization analyses, mask of circular ROIs for each channel was created. Masks of 2 channels were overlaid for double-colocalization analyses, and all three masks were overlaid for triple-colocalization analysis. Overlapped regions greater than 4 pixels were considered as positive colocalizations (shown in white) (5). Spines that contain NR1 and GluR2 on the analyzed dendrite are highlighted in white.