Alignment of Zn and Fe XRF maps of a DRG in FA and matching paraffin sections. (a), Zn XRF; (b), Fe XRF; (c) class-III-β-tubulin immunohistochemistry; (d), ferritin immunohistochemistry; (e), Zip14 immunohistochemistry. Low-power photographs of the stained sections were adjusted for size by reference to the mm scale on the XRF maps and rotated for optimal orientation. The illustrated DRG consisted of two portions of neural tissue that were identified by class-III-β-tubulin (c) and Zip14 reaction products (e). The region-of-interest containing the bulk of DRG neurons was outlined by interrupted lines. The outline was then transferred to the Zn and Fe XRF maps and the ferritin-stained section. Maps were segmented by vertical and horizontal lines placed at 0.5 mm intervals over the images to generate a grid. Within the maps, each square represented 0.25 mm2. At the edges, the squares were smaller. A single Zn or Fe signal was recorded as counts/10 sec from each square, and results of all signals were averaged for each metal. After subtracting background XRF, averaged counts were converted to μg Zn or Fe per ml PEG 1450. In the illustrated case of FA, Zn=4.8 μg/ml and Fe=33.4 μg/ml. The arrows in the two XRF maps indicate the location of Ti wires that are visible on the Zn and Fe XRF maps, respectively, due to minor contamination by these metals. Note strong Zn and Fe XRF arising from the capsule surrounding the neural portions of the DRG. These regions were effectively excluded from quantitative analysis by the illustrated alignment. Bars, 2 mm.