Take a pre-prepared HPF and FSF treated sapphire disc containing stable HeLa cells, wash it with PBS-A buffer for five minutes, and repeat this process three times. Transfer the sapphire discs to a 35-millimeter culture dish containing one milliliter of PBS-A buffer at room temperature. Replace the PBS-A buffer in the culture dish with one milliliter of reducing solution and incubate for one hour at room temperature.
Prepare the gold precursor in one milliliter of reducing solution and immediately mix by vortexing. Add 80 microliters of 500-millimolar D-Penicillamine in double distilled water to the gold precursor solution. And immediately vortex.
Replace the solution in the cultured dish with one milliliter of gold precursor solution and incubate for two hours at four degrees Celsius. Add 20 to 100 microliters of the freshly prepared sodium borohydride solution to the gold precursor solution, immediately shake to mix well, and incubate for five minutes at room temperature. Replace the solution with two milliliters of PBS-A buffer to stop the reaction.
Then prepare the epoxy resin mixture according to the manufacturer's instructions, transfer the sapphire discs to flat bottom embedding capsules, and infiltrate with resin for at least 30 minutes at room temperature. Polymerize the specimen at 60 degrees Celsius in an oven for at least 18 hours. Prepare ultra-thin sections of the polymerized specimen by trimming the specimen blocks carefully using a razor to expose the sapphire discs.
Dip the block tips with sapphire discs into liquid nitrogen for several seconds and thaw room temperature. Repeat the freeze-thaw action several times to detach the discs from the blocks. The EGFP-MTn-KDEL protein appeared as two to five-nanometer sized gold nanoparticles exclusively distributed in the peripheral ER lumen and in the perinuclear space of the nuclear envelope.
The well-preserved ultra structure enabled the single molecule identification of tagged proteins and facilitated the analysis of organelle interactions, such as ER mitochondria interactions. Nanoparticles of the Ost4-EGFP-MTn protein delineated the ER membrane in the outer membrane of the nuclear envelope. Likewise, the Mito-acGFP-MTn expressing cells exhibited specific labeling in the mitochondrial matrix.
