Combining immunofluorescence and MERFISH imaging in expanded samples. (a) Image of an expanded U-2 OS sample stained with MERFISH encoding probes for 129 RNA species, cadherin primary antibodies, oligo-conjugated secondary antibodies and visualized with a Cy5-conjugated complementary probe that detects the secondary antibodies. (b) Image of the same expanded sample visualized with a Cy5-labeled readout probe that detects one of the bits of the RNA barcodes at one focal plane for the white boxed region in (a). (c) Fluorescence images of all 8 rounds of two-color readout imaging for the orange boxed region in (b). (d) The average RNA copy numbers per cell for the 129 RNA species determined by MERFISH (~1,100 cells) vs. the abundances as determined by RNA-seq. The Pearson correlation coefficient (r) based on the log10 values of RNA abundances is 0.83. (e) The average RNA copy numbers per cell for the 129 RNA species determined by MERFISH in immunostained samples (~1,100 cells) vs. those in samples that are not immunostained (~1,000 cells). The Pearson correlation coefficient (r) is 0.95. The average ratio between the RNA copy numbers determined in the two experiments is 0.99 ± 0.03 (average ± s.e.m., N = 129 RNA species) and the median ratio is 0.94. (f) Image of unembedded U-2 OS cells stained with the same cadherin primary antibodies and oligo-conjugated secondary antibodies as well as a Cy5-conjugated complementary probe without any MERFISH RNA labeling and gel embedding. (g) Box plot of cadherin intensity per unit length of cell periphery (normalized to the actual length before expansion) in expanded MERFISH samples and in control immunostained samples that have not been subject to any MERFISH RNA labeling or gel embedding. The red lines indicate the median, the boxes mark the 25th to 75th percentiles, the whiskers extend to the most extreme data points not including outliers, and the outliers are plotted using the ‘+’ symbol. The P value is derived from a two-side Kolmogorov-Smirnov test (N = 20 areas each). The scale bars in (a-c) are as defined in Fig. 2. The white scale bar in (f) represents 10 µm.