Ixon ultra 888 emccd camera

Cells were fixed for 15 min with 4% PFA at room temperature and subsequently permeabilized in 70% EtOH overnight. Custom designed smFISH probes for Sox2 labeled with Quasar 670 (Stellaris® DesignReady FISH Probes, Cat# VSMF-3075–5-BS) were incubated with the samples for 16 hours at 30°C in hybridization buffer (100 mg/mL dextran sulfate, 25% formamide, 2X SSC, 1 mg/mL E.coli tRNA, 1 mM vanadyl ribonucleoside complex, 0.25 mg/mL BSA). Samples were washed twice for 30 min at 30°C with wash buffer (25% formamide, 2X SSC) containing DAPI (1 μg/mL, Sigma D9542). All solutions were prepared with RNAse-free water. Finally, the sections were mounted using ProlongGold (Life Technologies, P36930) and imaged two days later. Mounted samples were imaged on a Nikon Ti-Eclipse epifluorescence microscope equipped with an Andor iXON Ultra 888 EMCCD camera, using a 100X /1.45 Plan Apo Lambda oil objective (Nikon) and dedicated, custom-made fluorescence filter sets (Nikon). z-stacks with a distance of 0.3 μm between planes were collected. The number of Sox2 (mRNA) signals per cell was quantified using home-made MATLAB scripts.