Prepare a high pressure freezing or HPF machine, at least two hours before starting the experiment according to the manufacturer's instructions. Transfer Type A HPF aluminum carriers to a two-millimeter 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 disk from the HeLa cell culture dish. Touch the labeled surface with qualitative filter paper to remove the excess medium.
Mount the sapphire disk into the HPF specimen holder and quickly cap the disk 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 disk carrier assembly under liquid nitrogen in a foam cryo box. Prepare two milliliters of the freeze-substitution fixation or FSF solution one 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 disks and carrier assemblies into the container with frozen FSF solution one under liquid nitrogen.
Transfer this container to a pre-cooled automated freeze 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 disks from the carriers with pre-cooled tweezers, making sure that the marked sides of the sapphire disks are facing down. Keep the specimens at minus 90 degrees Celsius for an additional 8 to 10 hours before warming up to minus 60 degrees Celsius within three hours.
Replace the FSF solution one 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 two in a two-milliliter centrifuge tube at minus 30 degrees Celsius. Replace the acetone with FSF solution two and incubate at minus 30 degrees Celsius for three hours. Then replace FSF solution two in the container with pre-cooled acetone and incubate for 30 minutes at minus 30 degrees Celsius.
Repeat this acetone wash twice. Warm for minus 30 to 4 degrees Celsius within two hours. Replace the acetone with two milliliters of 0.2 molar HEPES buffer.
Change the buffer once and incubate at room temperature or four degrees Celsius overnight.
