Figure 2

EM distinction of QD565 for AT8 and QD655 for GFAP after gold enhancement and their LM counterparts on double immunolabeled section. (A) A section from the autopsied brain was double immunolabeled for AT8/QD565 (magenta) and GFAP/QD655 (green). (a) Extended focus image (EFI) of an NFT (open triangle) and astrocytes and its cross-sectional reconstruction (20 μm thick). Fluorescent signals from QD565 and QD655 were similarly intense throughout the entire depth of the section. (b) EFI of astrocytes on the same section at different site. An astrocyte enclosed in the rectangle was targeted for subsequent EM observation. (c,d) Bright-field LM counterpart of targets in (a,b) after gold enhancement and post-fixation. QD565 and QD655 labeled structures are darkened. (e–j) EM images corresponding to the LM images, after gold enhancement of the section. The areas enclosed by rectangles correspond to the images just below. While the filaments in NFT (e,g,i) was labeled with high contrast spherical QD565 (arrow), the glial fibers of the astrocyte (f,h,j) was labeled with rod-like QD655, which showed high contrast nanospheres deposited at the tips of the low contrast QD655 nanorods (filled triangle). The QD565 (in i, arrow) and the QD655 label (in j, filled triangle) are readily distinguishable from each other by their characteristic shapes after gold enhancement. Bars in a: 20 μm, b: 100 μm, c: 20 μm, d: 100 μm, e: 10 μm, f: 2 μm, g: 200 nm, h: 100 nm, i and j: 20 nm. (B) Another section of the same autopsied brain was labeled with ubiquitin/QD655 (magenta). A fluorescence image of a Lewy body (a, open triangle) were directly compared with bright-field LM image (b) and EM image (c–e) after gold enhancement. Loose and short ubiquitin-positive fibers of a Lewy body was labeled by rod-like QD655 (e, filled triangle), which showed high contrast nanosphere deposits at the tips of the nanorods. Bars in a and b: 20 μm, c: 5 μm, d: 100 nm, e: 20 nm.