Figure 4. Representative images of mediobasal hypothalamus (MBH) ROI and GFAP staining in autopsy samples.

Coronal brain slices that included the hypothalamus were imaged, sectioned, stained for glial fibrillary acidic protein (GFAP) via immunohistochemistry and digitally scanned. ROIs were placed on digital images (A, captured at 0.4X, scale bar represents 5 mm) and on a parametric map of T2 relaxation times derived from the multi-echo sequence for radiologic analysis (B, scale bar represents 5 mm). In both cases, the MBH ROI was placed adjacent to the 3^rd ventricle to encompass the location of the arcuate nucleus. Panels C-D show representative sections through the MBH from a brain slice with a high MBH T2 relaxation time and panels E-F are derived from a brain slice with a low MBH T2 relaxation time. Images in panels C and E depict immunostaining for GFAP (brown) and hematoxylin (blue) whereas panels D and F depict the identical region processed with digital color deconvolution software to measure staining intensity. Pseudocolors in D and F represent staining intensity (blue or white=no stain, yellow=low intensity and red=moderate or high intensity). Images in C-F captured at 40X; scale bar = 50 μm.