The video details a step-by-step protocol for high-pressure freezing and freeze-substitution fixation of biological samples. It includes preparation of aluminum carriers, use of sapphire disks, and controlled temperature incubations with various solutions to preserve cellular structures for further analysis.

high-pressure freezing (hpf), freeze-substitution fixation (fsf) and rehydration of sapphire disc-carrier assembly

Direct Synthesis of Gold Nanoparticles for Protein Localization Analysis

In the postgenomic era, the development of green fluorescent protein (GFP) as a single-molecule reporter for light microscopy has revolutionized the field of modern life science research1,2. For decades, electron microscopy (EM) has been a powerful tool for intuitively observing the cellular ultrastructure with nanoscale resolution3; however, the precise identification and localization of protein molecules remain challenging.
The most commonly used EM labeling technique is the immunoelectron microscopy (IEM) labeling technique, which is based on the antigen-antibody reaction. However, although many techniques have been developed in the field of IEM labeling, including pre-embedding IEM and post-embedding IEM (on resin sections or hydrated cryosections), it still suffers from low labeling efficiency (&#60;10%)4,5, which is related to the sample preparation and antibody quality. To overcome these limitations, developing genetically encoded tags has great application potential.
Two main types of EM tags have been thoroughly explored in recent years. One type is the DAB staining method, which utilizes tags such as APEX2 to oxidize 3,3&#39;-diaminobenzidine (DAB) to osmiophilic polymers for EM visualization6,7,8,9,10,11,12. It enables the labeling of high-abundance proteins in subcellular regions but is not suitable for single-molecule counting. The other type utilizes metal-binding proteins, such as ferritin13 and metallothionein (MT)14,15,16,17,18,19,20,21,22,23,24,25,26, to generate electron-dense metal deposits in situ for EM visualization. Only the latter has real potential for single-molecule visualization and counting. The molecular size of ferritin is too large (~450 kD) for it to be used as a promising tag, whereas the small size (~5 kD) of MT and its ability to bind various ions through its 20 cysteines have attracted great attention. Several labs have tried to label purified MT-fusion proteins or MT-expressing cells by incubating directly with Au+. These attempts have initially proved that MT tags can bind gold ions to form high-contrast signals, but none have really achieved the effective identification of individual proteins in cells, and they are not widely applicable14,15,16,17,18,19,20,21,22,23.
The ANSM-based AuNP synthesis technique, which involves synthesizing 2-6 nm-sized AuNPs directly on cysteine-rich tags (e.g., MT, the MT variants MTn and MT&#945;, AFP) as electron-dense labels for EM visualization, is the first reliable and applicable approach for protein labeling and single molecule detection in cells24,25,26. It allows for the specific synthesis of AuNPs on isolated tag fusion proteins and has achieved an unprecedented labeling efficiency in nonfixed or chemically fixed prokaryotic (E. coli) and eukaryotic (S. pombe) cells. However, implementing the same protocol in more advanced systems such as mammalian cells or even tissues involves additional challenges, such as the more complex intracellular redox homeostasis and more fragile cellular structure.
This study presents a clonable EM labeling technology, which combines the novel ANSM-based AuNP synthesis technique for labeling genetically encoded cysteine-rich tags (MT) with the HPF/FSF-rehydration-HPF/FSF sample preparation method, enables the unambiguous single-molecule identification of tagged proteins in the ER membrane, ER lumen, and mitochondrial matrix in HeLa cells. The current method combines the characteristics of high labeling efficiency, a high signal-to-noise ratio, single-molecule labeling, and strong universality, and this method has broad application prospects in life science research.

Author Spotlight: Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure  

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

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 protocols synthesizes in situ 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, yeast 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.

Watch this Scientific Journal Video about High-Pressure Freezing HPF, Freeze-Substitution Fixation FSF and Rehydration of Sapphire Disc-Carrier Assembly at JoVE.com

High-Pressure Freezing (HPF), Freeze-Substitution Fixation (FSF) and Rehydration of Sapphire Disc-Carrier Assembly  

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.

high-pressure-freezing-hpf-freeze-substitution-fixation-fsf

Research

JoVE Journal

Biology

203 vistas.  National Institute of Biological Sciences, Beijing. Prepare una máquina de congelación a alta presión o HPF, al menos dos horas antes de comenzar el experimento de acuerdo con las instrucciones del fabricante. Transfiera portadores de aluminio HPF tipo A a un tubo centrífugo de dos milímetros que contenga un mililitro de etanol y sonicarlos en un limpiador ultrasónico durante 10 minutos. Seque los portadores en una placa de Petri cubierta con papel de filtro cualitativo.Remoje los portadores prelimpiados en 1-hexadeceno. Use pinza...