Supplementary Figure 2: Resolution and penetration controls for Immuno-SABER.

(a) ~2.5 µm long lineplots (red) were made over the thin tubules to estimate the observed resolution. Yellow-boxed background regions were used to estimate the background for subtraction. (b) A typical line plot along a tubule and the Gaussian fit, where full-width-half-maximum (FWHM) was calculated as 371 nm. (c) Mean FWHM values were calculated for 30-45 lineplots from cells stained with Immuno-SABER or fluorophore-conjugated secondary antibodies samples and the distribution was displayed as a box plot. A similar calculation was performed for 200 nm fluorescent beads. Mean FWHM was not significantly different (p value is 0.360 for Immuno-SABER and 0.335 for conventional secondary antibody staining, two-sample t-test comparison to the bead sample). Box-plots are drawn with center line, median; box limits, upper and lower quartiles; whiskers, min and max values capped at 1.5× interquartile range. (d) Visualization of Collagen IV and Vimentin at multiple depths of the whole mount mouse retina shown in Fig. 2f. Selected confocal planes are shown. Vimentin stains the Muller cells and Collagen IV stains the blood vessels, both of which are localized predominantly in the segments from nerve fiber layer to outer plexiform layer (~100 µm) of the retina (Slijkerman et al., 2015). Hence it should be noted that, although the entire whole-mount retina is about ~180 μm, the target signal comes from roughly one half of the retina section, from the nerve fiber layer to the outer plexiform layer. Ref: Slijkerman, R.W. et al. The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies. Prog Retin Eye Res 48, 137-159 (2015).