MindsEye

Quantification of immunohistochemistry for Methods section

For the immunohistochemistry and X-Gal staining, cells expressing darkly-positive, nuclear staining were quantified with custom software (MindsEye, T. Houpt; Kwon et al. 2008). Regions were digitally-captured at 40x magnification on a Macintosh computer using an Olympus Provis AX-70 microscope with a Dage-MTI DC-330 CCD camera and Scion LG-3 framegrabber. Cells with dark nuclear staining were automatically detected and counted by the software across each image, based on the relative pixel darkness and circular symmetry of the nuclei relative to surrounding background tissue in the digitized image. To insure a consistent crtieria for the automatic counting, the same threshold parameters were used for all images from all treatments. Counting was restricted to the basolateral amygdala (BLA), central nucleus of the amygdala (CeA), or lateral amygdala (LA) as delineated by a hand-drawn outline. Bilateral cell counts were averaged for 6 sections of the amygdala for each mouse. The individual mean counts for each region were then averaged across mice within experimental groups. Significant effects across treatment groups were detected by one-way ANOVA and Neuman-Keuls post-hoc tests (Kaleidagraph, Synergy Software). All data are presented as the mean ± standard error of the mean.

Programming Notes

Description of Bruker MRI data file formats. We need to convert 2dseq file (a 3D block of data) into TIFF slices, probably in 3 different orientations (assuming TIFF can handle 16-bit grayscale. Otherwise may need to convert to RAW image files).