An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins
Transcript
Begin by washing a fixed heart section in a sodium cacodylate buffer that stabilizes the tissue structure. Immerse the section in osmium tetraoxide to stain the membrane lipids.
Treat with sodium acetate to maintain an optimum pH for staining.
Add uranyl acetate to stain biomolecules, providing better contrast during microscopy.
Dehydrate the tissue in increasing concentrations of ethanol followed by acetone.
Embed the tissue in a resin, obtain ultrathin sections, and transfer them onto a microscopy grid.
Introduce sodium meta-periodate to remove the osmium tetraoxide, unmasking the cellular protein.
The compound also oxidizes glycoprotein, creating reactive aldehyde groups, which are neutralized by glycine treatment.
Introduce a blocking buffer, preventing non-specific antibody binding.
Add primary antibodies specific to the target protein followed by biotinylated secondary antibodies.
Add streptavidin-conjugated quantum dots that bind to biotin.
Under an electron microscope, quantum dot-labeled regions exhibit higher contrast, aiding precise subcellular localization of the target protein.
