Supplementary Figure 7: Multicolor AbScale method applied to brain tissues from App^NL-F/NL-F mice and AD patients for 3D reconstruction

a) Optical components used for triple-color AbScale for immunostaining neuronal nuclei and Aβ plaques by a fluorescence stereomicroscope (Fig. 4). The excitation (dotted line) and emission (solid line) spectra of Alexa488, Cy3, and Cy5 are normalized and shown with the same colors as used in the main figures in CMYK mode. The transmission properties of the excitation and emission filters are illustrated by boxes. The filter sets used for Alexa488, Cy3, and Cy5 were ET-GFP, DSR, and ET-Cy5, respectively.

(b) Colocalization of pre-cut staining signals (Cy5-A60) with post-cut staining signals (Cy3-αNeuN) in the same section as shown in Fig. 4. Fluorescence images of Cy3 and Cy5 were acquired using a confocal microscope (FV1000) equipped with the optical components (indicated by the spectral graph), and shown in green and red, respectively, in RGB mode. Their colocalization was quantified by correlation analysis. The Pearson correlation coefficient (r) was calculated to be 0.969. Scale bars: 1 mm.

(c) Alexa488 and PP-BTA-1 for confocal microscopy (see Fig. 5a,b).

(d) Alexa488 and Texas Red for SPIM (see Fig. 5d).

(e) Alexa488 and Alexa546 for SPIM (see Fig. 6a–h).

(f) Alexa488, Alexa546, and Cy5 for SPIM (see Fig. 6i-r).

The excitation (dotted line) and emission (solid line) spectra are normalized and shown with the same colors as used in the main figures. The transmission properties of the emission filters are illustrated by boxes. ex, excitation; em, emission. The used laser lines are indicated by arrows.