Name: High-Pressure Freezing (HPF), Freeze-Substitution Fixation (FSF) and Rehydration of Sapphire Disc-Carrier Assembly
Uploaded: 2023-04-28T12:20:00
Description: Watch this Scientific Journal Video about High-Pressure Freezing HPF, Freeze-Substitution Fixation FSF and Rehydration of Sapphire Disc-Carrier Assembly at JoVE.com

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

Sintesi diretta di nanoparticelle d'oro per l'analisi della localizzazione delle proteine

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.

Autore in primo piano: Sintesi diretta di nanoparticelle d'oro EM-visibili nelle cellule per l'analisi della localizzazione delle proteine con ultrastruttura ben conservata  

Sintesi diretta di nanoparticelle d'oro EM-visibili nelle cellule per l'analisi della localizzazione delle proteine con ultrastruttura ben conservata

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  

Congelamento ad alta pressione (HPF), fissazione a sostituzione di freddo (FSF) e reidratazione del gruppo portadischi in zaffiro

Preparare una macchina di congelamento ad alta pressione o HPF, almeno due ore prima di iniziare l'esperimento secondo le istruzioni del produttore. Trasferire i supporti in alluminio HPF di tipo A in un tubo di centrifuga da due millimetri contenente un millilitro di etanolo e sonicarli in un pulitore ad ultrasuoni per 10 minuti. Asciugare i supporti in una capsula di Petri coperta con carta da filtro qualitativa.Immergere i vettori pre-puliti in 1-esadecene. Utilizzare una pinzetta fine per raccogliere un disco di zaffiro pre-preparato dal piatto di coltura cellulare HeLa. Toccare la superficie etichettata con carta da filtro qualitativa per rimuovere il mezzo in eccesso.Montare il disco di zaffiro nel supporto del campione HPF e tappare rapidamente il disco con un supporto in alluminio profondo 0,025 millimetri contenente 1-esadecene. Aspirare la soluzione in eccesso con carta da filtro qualitativa. Caricare il supporto del campione per il congelamento ad alta pressione.Scaricare il gruppo portadischi in zaffiro sotto azoto liquido in una scatola criogenica di schiuma. Preparare due millilitri di fissazione di congelamento-sostituzione o soluzione FSF uno in un contenitore rotondo di polipropilene da 20 millimetri in una cappa aspirante e congelarlo utilizzando azoto liquido. Utilizzare una coppia di pinzette pre-raffreddate per caricare i dischi di zaffiro e i gruppi di supporto nel contenitore con soluzione FSF congelata uno sotto azoto liquido.Trasferire questo contenitore in una camera della macchina di sostituzione del congelamento automatizzata pre-raffreddata per la lavorazione FSF a meno 90 gradi Celsius. Mantenere i campioni a meno 90 gradi Celsius per un'ora, quindi separare i dischi di zaffiro dai supporti con pinzette pre-raffreddate, assicurandosi che i lati marcati dei dischi di zaffiro siano rivolti verso il basso. Mantenere i campioni a meno 90 gradi Celsius per altre 8-10 ore prima di riscaldarsi fino a meno 60 gradi Celsius entro tre ore.Sostituire la soluzione FSF nel contenitore con acetone pre-raffreddato e incubare a meno 60 gradi Celsius per un'ora. Ripeti questo lavaggio con acetone due volte. Riscaldare fino a meno 30 gradi Celsius entro tre ore.Nel frattempo, preparare e pre-raffreddare due millilitri di soluzione FSF due in un tubo da centrifuga da due millilitri a meno 30 gradi Celsius. Sostituire l'acetone con la soluzione FSF due e incubare a meno 30 gradi Celsius per tre ore. Quindi sostituire la soluzione FSF due nel contenitore con acetone pre-raffreddato e incubare per 30 minuti a meno 30 gradi Celsius.Ripeti questo lavaggio con acetone due volte. Caldo per meno 30 a 4 gradi Celsius entro due ore. Sostituire l'acetone con due millilitri di tampone HEPES da 0,2 molari.Cambiare il tampone una volta e incubare a temperatura ambiente o quattro gradi Celsius durante la notte.

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