A novel technique for quantitative immunohistochemical imaging of various neurochemicals in a multiple-stained brain...

Here we describe a novel technique for comparative analysis of the distributions of various neurochemicals visualized using multiple immunohistochemistry in the same brain slice. As an example, the distributions of tyrosine hydroxylase, substance P and glutamate decarboxylase in coronal slices of rat brains were compared. Each slice was divided into approximately 220,000-300,000 microareas at 20-microm intervals, and the immunohistochemical intensities of the three substances in each microarea were analyzed independently using a brain mapping analyzer; a microphotometry system previously developed in our laboratory (Sutoo et al., J. Neurosci. Methods, 1998; 85: 161-73). No significant differences between the distribution of each substance were observed in single- and triple-labeled slices. We believe that this method will facilitate the investigation of the functions of the central nervous system and the disorders thereof in various diseases.