technique with a method to remove all the non-ER cytoplasmic
content of cells. This involves the selective permeabilization of the
plasma membrane using low levels of digitonin, which selectively
removes cytosolic contents from mammalian cells, including proteins, mRNA, and ribosomes that are not tethered to the ER
[14, 16, 20, 21]. Furthermore, when digitonin extraction is performed in a sucrose-containing buffer that preserves the integrity of
the ER [22], mRNA-ER interactions are maintained
Fig. 3 Single-molecule FISH analysis. The analysis of smFISH using the Spot Detection module in
NIS-Elements software. The top panel is an example of a raw image of smFISH of GAPDH mRNA in
digitonin-extracted U2OS cells using Stellaris smFISH probes conjugated to Quazar570. The bottom panel is
the same image where ROIs for the nucleus (magenta, ROI #1) and cell body (yellow, ROI #2) have been
selected to be analyzed using the Spot Detection Module, with each selected foci colored in magenta
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Jingze J. Wu and Alexander F. Palazzo