Single-Molecule FISH Labeling Protocol

Plated Wildtype S2 cells were first treated with arsenite (see above), fixed and labeled for endogenous FMR1 as described above in the IF section. After incubation with the secondary antibody, cells were washed 3 times with PBS and cells were post-fixed in 4% paraformaldehyde in PBS (pH 7.4) for 10 min. Following a washing 3 times in PBS, cells were further incubated for 5 min in 10% formamide (Thermofisher) in DEPC-treated water. They were then incubated overnight on a droplet containing one fluorescent smFISH probe (125 nM in 1% dextransulfate (D8906, Sigma Aldrich), 10% formamide (Thermofisher) in DEPC-treated water at 37°C) in a moistened chamber to avoid drying. Cells were washed 2 times for 30 min with 10% formamide (Thermofisher) in DEPC-treated water and mounted with Prolong antifade media (+DAPI) (ThermoFisher) on a microscope slide. All smFISH probes (Table S4) were labeled with Atto565 following (Gaspar et al., 2017 (link)). The DNA oligos were purchased from IDT. The TMR-oligo(dT)30x was also purchased from IDT.