Supplemental Figure 1. Colocalization of 6-CN-PiB fluorescence and either A40 (A-B) or
A42 (C-D) immunoreactive plaques in AD frontal cortex. Plaque load analysis shows a
correlation between percent cortical area labeled with 6-CN-PiB and A40 or A42
immunoreactive deposits (E). Scale bar = 75 m.
Supplemental Figure 2. Double-histofluorescence staining of a single section of an AD
entorhinal cortex, using BTA-1 (1 µM, A and C; blue, U filter) and X-34 (100 µM, B and D;
yellow-to-cyan, V filter) histofluorescence. Tissue autofluorescence (lipofuscin) is seen as
orange bleed-through signal in A and C. Boxed areas in A and B delineate areas of higher
magnification in C and D, respectively. Subpial diffuse A plaques, and a single cored plaque (C
and D, double-arrows) in lamina I are seen with both compounds. In lamina II, BTA-1 labels
only a few isolated structures (A, arrows), while X-34 also labels abundant NFTs and neuropil
threads (B). BTA-1 labeled structures inside layer II islands (C, arrows) appear brown/yellow
colored with X-34 histofluorescence (D, arrows), similar to the neighboring cored plaque (D,
double arrow); this makes them distinct from the surrounding NFTs that are seen as cyan-colored
with X-34 histofluorescence (B,D). Scale bar = 100 µm (A, B), 50 µm (C, D).
Supplemental Figure 3. Sequential tau IHC (A,B), 6-CN-PiB (C,D) and X-34 (E,F) histology in
a single hippocampal tissue section from an AD patient who had no A immunoreactive plaques
in this region. Each of the low power images (A,C,D) is a montage of 9 individual
photomicrographs; high power images (B,D,F) represent the boxed areas in corresponding lowpower images. There is an abundance of NFTs in the hippocampus (A,B). 6-CN-PiB signal is
very low (C,D), while X-34 fluorescence (E,F) prominently labels numerous NFTs and
dystrophic neurites in the same tissue section. Scale bar = 750 µm (A, C, E), 150 µm (B, D, F).
Supplemental Figure 4. (A) Scatchard analysis of [3H]PiB binding to fibrils formed from
synthetic A1-40 (squares and dashed lines) or A1-42 (circles and solid lines). The inset shows an
expansion of the high-affinity binding components. (B) Direct correlation of [3H]PiB binding
with total (combined A1-40 and A1-42) insoluble A levels determined by an ELISA in frozen
frontal and occipital cortex homogenates from six AD brains.
Supplemental Figure 5. Direct correlation between biochemical measurements of in vitro PiB
binding and region-matched analysis of total insoluble A (A1-40 plus A1-42) peptide levels in
fresh frozen tissue from the right hemisphere (A), and between plaque load quantifications of 6CN-PiB histochemistry and A (6E10) IHC in 25 tissue cubes dissected from the left hemisphere
(B), in the PiB PET AD subject postmortem.
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