ISH Profiling of Rat Cortex Figure 4. Cortical box method reveals the area-specific expression of RORbeta mRNAs. (A) Example of a cortical section of RORbeta ISH image processed for the cortical box standardization procedure. The mediodorsal end (MD), lateroventral end (LV), inner contour (IC), and outer contour (OC) were manually determined to select the part of the cortex for further processing. The selected cortical region was converted into a standard rectangle (a standardized cortical section). The intensity of the ISH signals was normalized and pseudocolored, so that the mean +1 SD becomes 0% and the mean +3 SD becomes 100% (see Materials and Methods for details). Scale bar, 2 mm. (B) Example of one series of coronal sections (from the Bregma distance of 22.1 to 26.2 mm, number 1 to 17) of RORbeta ISH (left). These images of cortical sections were standardized as displayed on the right side of the original images (representative). We performed the same processing for six series of such samples (right and left hemispheres from three rats) and averaged them. Note that the patterns of the representative and the average data are quite similar. We also performed the same procedure for the Nissl-stained samples and determined the cytoarchitectonic borders for primary somatosensory (Par1, yellow), visual (OC1, blue) and auditory (Te1, red) areas. (C) Layer distributions of RORbeta, ER81 and Nurr1 (left axis) as well as the Nissl-gray level index (GLI, right axis) from the pial surface (0% of cortical depth) to the cortex/white matter border (100%). Each line plot shows the average signal intensity at a given cortical depth. The entire cortical regions except the mediodorsal and laterocaudal 10% were used to calculate the average. Green, orange, blue and red lines represent RORbeta, ER81, Nurr1 ISHs and Nissl staining, respectively. (D) Conceptual figure to illustrate the construction of the cortical box. In this example, the layer 4 fraction (30–50% cortical depth) was extracted to demonstrate the area distribution pattern of RORbeta mRNA in a two-dimensional map. doi:10.1371/journal.pone.0003266.g004 rats is shown. Note that the pattern of a single set of sample (‘‘Representative’’) was very similar to that of the average. The characteristic expression pattern of RORbeta mRNA is therefore reproducibly captured across different animals. In the final step of image processing, we arrayed the seventeen cortical rectangles, so that the posterior cortex is three-dimen- sionally reproduced as a box (Fig. 4D). By this procedure, the expression data are now mapped onto a standardized cortical box, PLoS ONE | www.plosone.org 4 September 2008 | Volume 3 | Issue 9 | e3266