Visualizing Subcellular Localization of a Protein in the Heart using Quantum Dots-Mediated Immuno-Labeling followed by Transmission Electron Microscopy.

The subcellular localization is critical to delineating proper function and determining the molecular mechanisms of a particular protein. Several qualitative and quantitative techniques are used to determine the subcellular localization of proteins. One of the emerging techniques in determining the subcellular localization of a protein is quantum dots (QD)-mediated immunolabeling of a protein followed by imaging them with transmission electron microscopy (TEM). QD is a semiconductor nanocrystal with a dual property of crystalline structure and high electron density, which makes them applicable to electron microscopy. This current method visualized the subcellular localization of Sigma 1 receptor (Sigmar1) protein using QD-TEM in the heart tissue at ultrastructural level. Small cubes of the heart tissue sections from a wild-type mouse were fixed in 3% glutaraldehyde, subsequently osmicated, stained with uranyl acetate, followed by sequential dehydration with ethanol and acetone. These dehydrated heart tissue sections were embedded in low-viscosity epoxy resins, cut into thin sections of 500 nm thickness, put on the grid, and subsequently subjected to antigen unmasking with 5% sodium metaperiodate, followed by quenching of the residual aldehydes with glycine. The tissues were blocked, followed by sequential incubation in primary antibody, biotinylated secondary antibody, and streptavidin-conjugated QD. These stained sections were blot dried and imaged at high magnification using TEM. The QD-TEM technique allowed the visualization of Sigmar1 protein's subcellular localization at the ultrastructural level in the heart. These techniques can be used to visualize the presence of any protein and subcellular localization in any organ system.