Protein detection and localization are essential in biological research. Our protocol describes a novel EM labeling technology using genetically encoded tags, which allows single molecule visualization in situ with well-preserved ultrastructure. It is crucial to avoid over fixation.
The labeling method is based on the activity of cysteines in the tags, which are highly sensitive to aldehyde cross-linking. Our protocol avoids use of aldehyde fixatives, and preserves the tag activity and ultrastructure at the same time. EM labeling has always been challenging, suffering from poor morphology or inefficient labeling.
Our protocol synthesizes residual nanoparticles directly on individual proteins to provide clear and precise localization of the target proteins with a high signal to noise ratio, high efficiency and specificity. We have so far achieved excellent labeling in isolated proteins, E.coli cells, Easter cells, and HeLa cells. We will explore and optimize the method for application to tissue samples.
Combining the prevailing cryo-FIB and cryo-EM technologies will allow us to address important biological questions. Transfer three millimeters by 0.16 millimeters sapphire discs to a two milliliter centrifuge tube containing one milliliter ethanol and sonicate it in an ultrasonic cleaner for 10 minutes. Burn each sapphire disc in an alcohol lamp flame until there are no visible deposits on the surface.
Label one side of each sapphire disc with a number using a solvent-resistant pen. Place the labeled sapphire discs on the bottom of a 35-millimeter culture dish with the labeled side facing up. Sterilize the cell culture dish with sapphire discs under ultraviolet light for 30 minutes.
Flip the sapphire discs with tweezers ensuring that the labeled side is facing down and sterilize under ultraviolet light for an additional 30 minutes. Seed the stable HeLa cell lines expressing MTN tags on the prepared sapphire discs. And grow to 80 to 90%confluency in a cell incubator.
Prepare a high pressure freezing or HPF machine at least two hours before starting the experiment according to the manufacturers instructions. Transfer type A HPF aluminum carriers to a two milliliter centrifuge tube containing one milliliter ethanol and sonicate them in an ultrasonic cleaner for 10 minutes. Dry the carriers in a Petri dish covered with qualitative filter paper.
Soak the pre-cleaned carriers in 1-hexadecene. Use fine tweezers to pick up a pre-prepared sapphire disc from the HeLa cell culture dish. Touch the labeled surface with qualitative filter paper to remove the excess medium.
Mount the sapphire disc into the HPF specimen holder and quickly cap the disc with a 0.025 millimeters deep aluminum carrier containing 1-hexadecene. Aspirate excess solution with qualitative filter paper. Load the specimen holder for high pressure freezing.
Unload the sapphire disc carrier assembly under liquid nitrogen in a foam cryo box. Prepare two milliliters of the free substitution fixation or FSF Solution 1 in a 20 millimeter round polypropylene container in a fume hood and freeze it using liquid nitrogen. Use a pair of pre-cooled tweezers to load the sapphire discs and carrier assemblies into the container with frozen FSF Solution 1 under liquid nitrogen.
Transfer this container to a pre-cooled automated free substitution machine chamber for FSF processing at minus 90 degrees Celsius. Keep the specimens at minus 90 degrees Celsius for one hour. Then separate the sapphire discs from the carriers with pre-cool tweezers, making sure that the marked sides of the sapphire discs are facing down.
Keep the specimens at minus 90 degrees Celsius for an additional eight to 10 hours before warming up to minus 60 degrees Celsius within three hours. Replace the FSF Solution 1 in the container with pre-cooled acetone and incubate at minus 60 degrees Celsius for one hour. Repeat this acetone wash twice.
Warm up to minus 30 degrees Celsius within three hours. Meanwhile, prepare and pre-cool two milliliters of FSF Solution 2 in a two milliliter centrifuge tube at minus 30 degrees Celsius. Replace the acetone with FSF Solution 2 and incubate at minus 30 degrees Celsius for three hours.
Then, replace FSF Solution 2 in the container with pre-cooled acetone and incubate for 30 minutes at minus 30 degrees Celsius. Repeat this acetone wash twice. Warm from minus 30 to four degrees Celsius within two hours.
Replace the acetone with two milliliters of 0.2 molar heaps buffer. Change the buffer once, and incubate at room temperature or four degrees Celsius overnight. Take a pre-prepared HPF and FSF treated sapphire disc containing stable HeLA cells, wash it with PBSA buffer for five minutes, and repeat this process three times.
Transfer the sapphire discs to a 35 millimeter culture dish containing one milliliter of PBSA buffer at room temperature. Replace the PBSA 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 depenicillamine in double distilled water to the gold precursor solution, and immediately vortex. Replace the solution in the culture 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 PBSA 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 at 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 ultrastructure 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.
