GFP-ARER:PRL-HeLa Fluorescent Imaging

GFP-ARER:PRL-HeLa were fixed in 4% formaldehyde in ribonuclease-free phosphate-buffered saline for 30 minutes, washed in PBS, and then permeabilized with 70% ethanol in ribonuclease-free water for a minimum of 1 hour at 4°C. Cells were washed in 1 mL of wash buffer A (LGC Biosearch Technologies, Novato, CA) containing 10% v/v formamide followed by hybridization with custom made dsRED2 RNA probes^{18 (link),19 (link),24 (link)} (Stellaris® probes; LGC Biosearch Technologies) diluted 1:500 (50 nM) in hybridization buffer (1 g dextran sulfate, 1 mL 20X saline sodium citrate (SSC) buffer and 1 mL formamide in 8 mL of nuclease-free water) overnight at 37C. After hybridization, cells were washed twice (15 min at 37C) with wash buffer A then with wash buffer B (LGC Biosearch Technologies) for 30 minutes at 37C and then stained with DAPI for 10 minutes at 37C. After DAPI labeling the cells were washed x1 with Dulbecco’s PBS, then stored and imaged in Dulbecco’s PBS + 0.02% Sodium azide.