G2133 10ku

The primary probe hybridization buffer consisted of 133 mg/mL high-molecular-weight dextran sulfate (Calbiochem #3710–50GM), 2X saline-sodium citrate (SSC) (IBI Scientific #IB72010), and 66% formamide (Bio Basic #FB0211–500) in ultrapure water. The 55% wash buffer was comprised of 2X SSC, 55% formamide, and 1% Triton-X (Sigma-Aldrich #10789704001) in ultrapure water. The secondary probe hybridization buffer consisted of 2X SSC, 16% ethylene carbonate (Sigma-Aldrich #E26258–500G), and 167 mg/mL of high-molecular-weight dextran sulfate in ultrapure water. The ethylene carbonate was first melted at 50°C for 30–60 minutes. The 10% wash buffer was comprised of 2X SSC, 10% formamide, and 1% Triton-X in ultrapure water. The imaging buffer base consisted of 0.072M Tris HCl (pH 8) (RPI #T60050–1000), 0.43 M NaCl (Fisher #MK-7581–500), and 3 mM Trolox (Sigma-Aldrich #238813) in ultrapure water. The anti-bleaching buffer was comprised of 70% imaging buffer base, 2X SSC, 1% catalase (10X dilution of stock) (Sigma #C3155), 0.005 mg/mL glucose oxidase (Sigma-Aldrich #G2133–10KU), and 0.08% D-glucose (Sigma #G7528) in ultrapure water.

Cells were grown on coverslips and fixed for 20 min in 4% PFA and overnight with 70% ethanol at 4°C. The next day, cells were washed with PBS and treated for 2.5 min with 0.5% Triton X-100. Cells were washed with PBS and incubated for 10 min in 15% formamide (in 4% SSC; Bio-Lab Ltd, Jerusalem, Israel). Cells were hybridized overnight at 37°C in 15% formamide with a specific Cy3-labeled oligo(dT) DNA probe (∼10 ng probe, 50mer). The next day, cells were washed twice with 15% formamide for 15 min and then immunofluorescence was performed after the RNA FISH using the standard protocol described above. Nuclei were counterstained with Hoechst 33342, and coverslips were mounted in mounting medium.
smFISH experiments with Stellaris probes (Biosearch Technologies) were performed according to the manufacturer's adherent cell protocol for MKI67 and IPO7 mRNA, as previously described (Sheinberger et al., 2017 (link)). NORAD probes were from Igor Ulitsky (Weizmann Institute of Science, Rehovot, Israel; Zuckerman et al., 2020 (link)). To reduce photobleaching, cells were submerged in GLOX buffer (pH 8, 10 mM Tris-HCl, 2× SSC, 0.4% glucose) supplemented with 3.7 ng of glucose oxidase (Sigma, G2133-10KU) and 1 µl catalase (Sigma, 3515) prior to imaging (Raj et al., 2008 (link); Yildiz et al., 2003 (link)).

In this research, we used oxygen scavenger buffer (Glox-buffer). We prepared a glucose stock solution (300 mM glucose, 50 mM Tris, 10 mM NaCl dissolved in Milli-Q H₂O) and stored it at 4°. The final concentration of each ingredients are 1.25 mg/ml catalase (Sigma, C40-100MG), 1 mg/ml glucose-oxidase (Sigma, G2133-10KU), and 50~150 mM MEA (Sigma, 30070-10G) diluted in glucose stock. We adjusted the blinking density by adjusting the concentration of MEA.