FISH was performed as described previously (Rouquette et al., 2005 (link)) using a 5’ Cy3-labeled 5’ITS1 probe (5’-CCTCGCCCTCCGGGCTCCGTTAATGATC -3’; Microsynth).
Cells grown on coverslips were fixed in 4% PFA/PBS for 30 min at room temperature. After two rinses with PBS, cells were permeabilized by overnight incubation with 70% ethanol. Cells were rehydrated by two incubations for 5 min in 10% formamide in 2X SSC (30 mM tri-sodium citrate dihydrate pH 7.0, 300 mM NaCl). Hybridization was performed for 4 hr at 37°C in 10% formamide in 2X SSC supplemented with 10% dextran sulfate, 10 mM ribonucleoside vanadyl complexes (Sigma-Aldrich, cat#R-3380), 50 μg/ml BSA (Sigma-Aldrich, cat#B-2518), 0.5 μg/μl tRNA (Sigma-Aldrich, cat#R-1753), and 0.5 ng/μl Cy3-5’ITS1 probe. Subsequently, cells were washed twice for 30 min with 10% formamide in 2X SSC before washing for 5 min with PBS and mounting of the coverslips using Vectashield (Vector Laboratories). After imaging by confocal microscopy, the cytoplasmic signal was measured with CellProfiler version 4.2.0 (McQuin et al., 2018 (link)) and quantified with the GraphPad Prism software.
Cells grown on coverslips were fixed in 4% PFA/PBS for 30 min at room temperature. After two rinses with PBS, cells were permeabilized by overnight incubation with 70% ethanol. Cells were rehydrated by two incubations for 5 min in 10% formamide in 2X SSC (30 mM tri-sodium citrate dihydrate pH 7.0, 300 mM NaCl). Hybridization was performed for 4 hr at 37°C in 10% formamide in 2X SSC supplemented with 10% dextran sulfate, 10 mM ribonucleoside vanadyl complexes (Sigma-Aldrich, cat#R-3380), 50 μg/ml BSA (Sigma-Aldrich, cat#B-2518), 0.5 μg/μl tRNA (Sigma-Aldrich, cat#R-1753), and 0.5 ng/μl Cy3-5’ITS1 probe. Subsequently, cells were washed twice for 30 min with 10% formamide in 2X SSC before washing for 5 min with PBS and mounting of the coverslips using Vectashield (Vector Laboratories). After imaging by confocal microscopy, the cytoplasmic signal was measured with CellProfiler version 4.2.0 (McQuin et al., 2018 (link)) and quantified with the GraphPad Prism software.
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