We designed oligonucleotide probe sets using the Stellaris probe designer (Biosearch Technologies) and ordered them with an amine group on the 3′ end (sequences available in Supplementary Data 3 ). We pooled the oligonucleotides for each probe set and coupled them to either Cy3 (GE Healthcare), Alexa 594 (Life Technologies), Atto647N or Atto 700 (Atto-Tec). We performed RNA FISH as previously described30 (link) for each of the cycles of hybridization. We first fixed cells with formaldehyde and permeabilized with 70% ethanol. We next washed once with wash buffer (containing 10% formamide and 2X SSC) and then applied hybridization buffer (containing 10% formamide, 10% dextran sulfate, and 2X SSC) with the specified pool of RNA FISH probes. We hybridized for 6–12 hours and then washed 2 times for 30 minutes with wash buffer.
After imaging, we applied 60% formamide with 2X SSC for 15 minutes on a heat plate kept at 37℃. We then washed the sample 3 times with 1X PBS for 15 minutes also at 37℃ to remove residual formamide, which we have found can inhibit further hybridizations. Lastly, we washed once with wash buffer to remove residual 1X PBS and prepare the samples for another RNA FISH hybridization.
After imaging, we applied 60% formamide with 2X SSC for 15 minutes on a heat plate kept at 37℃. We then washed the sample 3 times with 1X PBS for 15 minutes also at 37℃ to remove residual formamide, which we have found can inhibit further hybridizations. Lastly, we washed once with wash buffer to remove residual 1X PBS and prepare the samples for another RNA FISH hybridization.