Fig. 3
From: Protocol optimization improves the performance of multiplexed RNA imaging
Improved Dye Brightness and Stability with Revised Readout Reagent Buffers. (A, B) Images of U-2 OS cells stained with encoding probes that target 130 RNAs and with the first Cy5-labeled readout probe after the first (left) or second (right) exposure of the same sample region in an imaging buffer with a base of saline sodium citrate (SSC) pH 7 (A) or a Tris-HCl pH 8 (B). The top and bottom rows represent images from the first (top) or 80th (bottom) field of view (FOV) imaged immediately after imaging buffer was flown into the sample. (C, D) Precent loss in single-molecule brightness in the second imaged frame relative to that of the first imaged frame for Cy5 (C) or AF750 (D) imaged in imaging buffers with bases of SSC pH 7, Tris pH 8, TAPS pH 8, or Glycine (Gly.) pH 9 (Methods). (E) Average first-frame single-molecule signal brightness measured for the Cy5 and AF750 channels across all FOVs of the measurements in (A) and (B). For A and B: Gray: mRNA signal. Scale bars: 20 μm. For C, D, and E: Bars or solid lines represent averages and shaded areas or error bars represent standard deviation across the average of plotted quantities seen between two replicates.
