2xssc

RNA FISH was performed with small changes according to [40 ]. Oocytes were fixed for 10 min in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS with 40 units/20 μl of RNAseOut (Invitrogen), then mounted to pre-coated Lab-Tek II Chamber Slide System slides using 80% methanol pre-frozen to -20°C. Oocytes were washed in Wash Buffer A (Biosearch Technologies) and incubated overnight at 30°C in hybridization buffer (Biosearch Technologies) with 75 nM oligo-d(T) probe (Biotech Generi); Neat2 CalFluorRed610 (Biosearch Technologies); Dazl (Biosearch Technologies) and β-actin labelled with Cy5 (Biotech Generi); GFP CalFluorRed635 (Biosearch Technologies) with 75 nM (protected from light). Oocytes were then washed 3x in Wash Buffer A and 2x in 2xSSC (Sigma Aldrich). For visualization of chromatin structure the oocytes were incubated 1 min with 10 nM DAPI (Sigma Aldrich) in 2xSSC; then washed 1x with 2xSSC and scanned in 2xSSC. For negative control RNase A (Ambion) was used for 2 h at 37°C after the permeabilization step. Forty-five oocytes and embryos was analyzed using ImageJ/FIJI (http://rsbweb.nih.gov/ij/) for quantification of fluorescence intensity in the cytoplasm and the nucleus.

Astrocytes on glass coverslips were fixed with 4% paraformaldehyde (Agar Scientific) for 15 min at room temperature followed by permeabilization in 70% ethanol at 4°C overnight. Cells were then re‐hydrated in 50% formamide (Sigma)/2x SSC (Sigma) for 10 min at room temperature and blocked in hybridization buffer (50% formamide (Sigma), 2×SSC (Sigma), 10% Dextran Sulfate (Millipore), 1 mg/ml Yeast tRNA (Invitrogen) and 1 mg/ml Salmon Sperm DNA (Invitrogen)) for 30 min at 45°C. 50 ng of an Alexa Fluor® 546‐conjugated (GGCCCC)₄ probe (IDT) diluted in the hybridization buffer was applied on cells for 2 hours at 45°C in a humidified chamber. After the hybridization, cells were washed twice with 50% formamide/2× SSC for 30 min at 45°C and then once with 2× SSC for 30 min at room temperature. After another three washes with PBS at room temperature, immunofluorescence imaging was performed as described previously.
As controls, cells were treated with either 3 U/ml DNase (Life Technologies) or 100 μg/ml RNase (Sigma) diluted in 2x SSC for 1 hour at 37°C prior to the hybridization step. In addition, an anti‐sense RNA probe against the CCTG repeat expansion was also applied on cells to assess the specificity of the (GGCCCC)₄ probe.