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. Author manuscript; available in PMC: 2016 Mar 2.
Published in final edited form as: Chembiochem. 2015 Feb 6;16(4):611–617. doi: 10.1002/cbic.201402715

Figure 3. Using eChol as an imaging probe for microscopic detection of Chol in cells.

Figure 3

(A) NIH-3T3 cells incubated for 12 hours with or without eChol (20 μM from DMSO stock), were stained by CuAAC with fluorescein-azide and by Hoechst, and were then imaged by fluorescence microscopy. The eChol staining pattern is characteristic of incorporation into various cellular membranes.

(B) Higher magnification view of a cell labeled with eChol and stained with fluorescein-azide.

(C) Subcellular distribution of eChol. NIH-3T3 cells were transiently transfected with plasmids expressing red fluorescent protein (RFP) fusions targeted to various cellular membranes: plasma membrane (pmCherry-CAAX plasmid), endoplasmic reticulum (ER) (pmCherry-Sec61β plasmid) and mitochondria (pDsRed-Mito plasmid). The cells were then labeled with eChol and stained with fluorescein-azide as in (A). Left panels: RFP images; middle panels: eChol images; right panels: overlay of RFP (red) and eChol (green). EChol appears enriched in mitochondria relative to the ER (compare bottom and middle panels), consistent with the low levels of Chol normally found in ER. EChol also co-localizes with the plasma membrane marker (top panels).

(D) As in (C), but showing a high magnification view of eChol co-localization with the plasma membrane marker, mCherry-CAAX. Left panel: mCherry-CAAX image; right panel: eChol image. The two cells at the bottom of the micrographs express mCherry-CAAX, while the two top cells do not.