Supplementary Figure 4: Accessibility, concatemer length, and quantitativeness controls for Immuno-SABER.

(a) Experiment design: HeLa cell preparations were stained with anti-Lamin B antibodies and oligo-conjugated secondary antibodies. Concatemers of different sizes (short: 350 nt, medium: 450 nt, long: 700 nt; length estimations are based on the gel run with respect to dsDNA ladder) were then hybridized to the antibodies. For this case, concatemers that contain an extra orthogonal binding site (for i.27*, as baseline imager) before the primer domain (p.28) were utilized. Samples were imaged first with only the baseline imager, then with the amplifier imager (i.28*). Imaging was done at 100× and 22-25 plane z stacks were acquired by an epifluorescence microscope. (b) Nuclei were segmented based on DAPI and mean Lamin B fluorescence intensity per nuclear pixel was calculated. n = 23-45 cells (individual sample sizes are written in parenthesis above each bar). Error bars, s.e.m. (c) For reference, the same target was also stained with commercial fluorophore-conjugated secondaries that on average bear 5× Alexa647 fluorophores and the fluorescence was measured the same way. (d) Representative images show the maximum projections for DAPI and Lamin B staining (Alexa647) for baseline and amplification conditions. The intensity scaling for each row is note on the left-hand side. (e) Secondary antibody-fluorophore images are shown for comparison. (f) Branching experiment design: A separate set of cells were similarly imaged with the baseline imager after hybridization of short (350 nt) or long primary concatemers (700 nt). This was followed by hybridization of secondary concatemers (short: 250 nt, long: 450 nt). Secondary concatemers also contained an orthogonal binding site (for i.30*) before the primer domain (p.25*). Baseline for branching (post-linear) was imaged by i.30*, followed by branched amplification with i.25*. (g) Lamin B fluorescence intensity per nuclear pixel was calculated as in b and is shown with the bar plot (left axis) overlaid with the scatter plot showing the distribution in the dataset. The dot plot above (magenta, right axis) shows the coefficient of variation for all the conditions. Note that the imagers bind dimers of primer units, whereas branches bind trimers for higher stability during exchange rounds. Therefore compared to the i.28* staining in previous panels, i.30* is expected to yield 1.5-fold lower signal (blue bars in b and g). (h) Representative images show the maximum projections corresponding to the conditions in g. All images are acquired under comparable conditions, and are displayed at the given intensity scaling for each amplification level.