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

直接合成金纳米颗粒用于蛋白质定位分析

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.

作者聚焦：在细胞中直接合成 EM-Visible 金纳米颗粒，用于具有保存完好的超微结构的蛋白质定位分析  

在细胞中直接合成EM-可见金纳米颗粒，用于蛋白质定位分析，具有保存完好的超微结构

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  

蓝宝石盘载体组件的高压冷冻 （HPF）、冷冻替代固定 （FSF） 和再水化

根据制造商的说明准备高压冷冻或HPF机器，至少在开始实验前两个小时。将A型HPF铝载体转移到含有1毫升乙醇的2毫米离心管中，并在超声波清洗机中超声处理10分钟。在覆盖有定性滤纸的培养皿中干燥载体。将预先清洁的载体浸泡在1-十六碳烯中。使用细镊子从HeLa细胞培养皿中取出预先准备好的蓝宝石盘。用定性滤纸触摸标记的表面以去除多余的介质。将蓝宝石圆盘安装到 HPF 样品支架中，并用含有 1-十六烯的 0.025 毫米深铝载体快速盖住圆盘。用定性滤纸吸出过量溶液。装载试样架进行高压冷冻。在液氮下将蓝宝石盘载体组件卸载到泡沫冷冻盒中。在通风橱中的20毫米圆形聚丙烯容器中准备两毫升冷冻替代固定或FSF溶液，并使用液氮冷冻。使用一对预冷的镊子将蓝宝石盘和载体组件装入装有液氮冷冻 FSF 溶液的容器中。将此容器转移到预冷的自动冷冻替代机室中，以便在零下90摄氏度下进行FSF处理。将标本保持在零下 90 摄氏度一小时，然后用预冷镊子将蓝宝石盘与载体分开，确保蓝宝石盘的标记侧面朝下。将标本在零下 90 摄氏度下再保温 8 到 10 小时，然后在三小时内升温到零下 60 摄氏度。用预冷的丙酮替换容器中的FSF溶液，并在零下60摄氏度下孵育一小时。重复这种丙酮洗涤两次。在三小时内预热至零下 30 摄氏度。同时，在零下30摄氏度的两毫升离心管中制备并预冷两毫升FSF溶液。用FSF溶液二代替丙酮，并在零下30摄氏度下孵育3小时。然后用预冷的丙酮替换容器中的FSF溶液二，并在零下30摄氏度下孵育30分钟。重复这种丙酮洗涤两次。在两小时内加热零下 30 到 4 摄氏度。用两毫升0.2摩尔HEPES缓冲液代替丙酮。更换缓冲液一次，并在室温或四摄氏度下孵育过夜。

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

Research

JoVE Journal

Biology

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

228 次观看.  National Institute of Biological Sciences, Beijing. 根据制造商的说明准备高压冷冻或HPF机器，至少在开始实验前两个小时。将A型HPF铝载体转移到含有1毫升乙醇的2毫米离心管中，并在超声波清洗机中超声处理10分钟。在覆盖有定性滤纸的培养皿中干燥载体。将预先清洁的载体浸泡在1-十六碳烯中。使用细镊子从HeLa细胞培养皿中取出预先准备好的蓝宝石盘。用定性滤纸触摸标记的表面以去除多余的介质。将蓝宝石圆?...

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

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

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various ...

科研

生物学

﻿Preparing Sapphire Discs for Cell Culture

Preparing Sapphire Discs for Cell Culture  

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.

制备用于细胞培养的蓝宝石盘

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.

Gold Nanoparticle Synthesis for Mammalian Cells Using Sapphire Discs

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.