Fig. 2
From: Protocol optimization improves the performance of multiplexed RNA imaging
Long-Term Stability of MERFISH Readout Reagents. (A) Images of U-2 OS cells stained with encoding probes that target 130 RNAs and the first Cy5- or AF750-labeled readouts in the first imaging round (left), the fourth Cy5- or AF750-labeled readouts in the fourth imaging round (middle), or the eighth Cy5- or AF750-labeled readout probes in the eighth imaging round (right). The titles list the approximate time since the readout reagents were made. (B) Single-molecule signal brightness for the Cy5 or AF750 channels in each of the hybridization and imaging rounds with the approximate age of the readout reagents. (C) Images of U-2 OS cells stained with encoding probes that target FLNA and with a Cy5-labeled readout probe after 1, 10, or 500 image exposures with an imaging buffer prepared freshly (top row) or prepared and aged for 7 days at room temperature protected from oxygen (bottom). (D) Single-molecule signal brightness measured for the Cy5 channel versus the total exposure time for samples as in (C). Intensity is measured in precent brightness decrease relative to that observed in the first image. (E) Images of U-2 OS cells stained with encoding probes that target 130 RNAs and with Cy5-labeled (top) or AF750-labeled (bottom) readout probes in hybridization reagents that were prepared freshly (left) or prepared and aged in the dark but exposed to the room environment for 7 days covered (right) or uncovered (middle). Where denoted the contrast has been increased by 2-fold. (F) RNA copy number per cell for samples prepared as in (E). ‘No incubation’ indicates reagents prepared freshly, ‘Open Air’ indicates reagents aged in the dark but exposed to the room environment, ‘Parafilm’ and ‘Mineral Oil’ indicate reagents aged in the dark but protected from the room environment with parafilm or a layer of mineral oil, respectively. For A, C, E: Gray: mRNA signal. Blue: DAPI. Scale bars: 20 μm. For B, D, F: Bars and error bars or shaded regions represent the average and standard deviation derived from the plotted quantity from each of at least two replicates.
