Prolong antifade media dapi

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 (Suppl Table S4) were labeled with Atto565 following (32) . The DNA oligos were purchased from IDT. The TMR-oligo(dT)30x was also purchased from IDT.

After treatments, S2 cells and dissociated brain cells were fixed with 4 % paraformaldehyde (Sigma Aldrich) in PBS (pH 7.4) for 20 min. Cells were then washed 3 times with PBS and subsequently quenched by incubation in 50 mM NH4Cl in PBS for 5 min. Followed by permeabilization with 0.11 % Triton-X for 5 min. Hereafter, cells were washed 3 times in PBS and blocked in PBS supplemented with 0.5 % fish skin gelatin (Sigma Aldrich) for 20 min. Cells were then incubated with the primary antibody (in blocking buffer) for 25 min, washed 3 times with blocking buffer and incubated with the secondary antibody (in blocking buffer) coupled to a fluorescent dye for 20 min. Cells on the coverslip were washed 2 times in milliQ and dried for 3 min on a tissue with cells facing up. Finally, each coverslip with cells was mounted with Prolong antifade media (+DAPI)(ThermoFisher) on a microscope slide.