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Cells grown on 1.5 circular glass coverslips were washed with ice-cold 1X PBS and fixed in 4% paraformaldehyde for 10 min at room temperature 38
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Because inhomogeneous cellular distribution of reacted and unreacted probes may bias the ratiometric calculations, especially at the subcellular resolution, we opted for RT instead of RT-NH 2 to achieve high-quality quantitative results in living cells
