A simple and reliable quick-freezing/freeze-fracturing procedure

Fujimoto, Kazushi; Noda, Tetsuji; Fujimoto, Toyoshi

doi:10.1007/s004180050091

Cited by 11 publications

(6 citation statements)

References 6 publications

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“…SDS-digested freeze-fracture labeling (SDS-FRL) combines freeze-fracture electron microscopy with colloidal gold immunocytochemistry, which has proved to be a powerful technique for visual identification of membrane topology and the spatial distribution of various components of eukaryotic biomembranes (e.g., Fujimoto et al 1997;Dunia et al 1998;Takizawa 1999).…”

Section: Discussionmentioning

confidence: 99%

A New Method to Visualize the Helicobacter pylori-associated Lewis^b-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

Petersson

Larsson

Mahdavi

et al. 2000

J Histochem Cytochem.

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(JM,TB) S U M M A R Y Freeze-fracture replica labeling has become a versatile tool to visualize both membrane components and other cell structures using SDS-replica cleaning before specific immunogold labeling of proteins or lipids. We report here for the first time the adoption and optimization of the method to studies of bacterial envelopes, as applied to structural analysis of the distribution of the unique BabA-adhesin of the gastric pathogen Helicobacter pylori. BabA is important for bacterial adherence to the human epithelial cell lining of the stomach. The adhesin was found to be distributed all over the bacterial cell surfaces. Our results suggest that the SDS-replica labeling allows assessment of protein localization to distinct cell compartments and analysis of co-localization with neighboring membrane structures.

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Section: Discussionmentioning

confidence: 99%

A New Method to Visualize the Helicobacter pylori-associated Lewis^b-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

Petersson

Larsson

Mahdavi

et al. 2000

J Histochem Cytochem.

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“…Platinum/carbon shadowing was performed using an angle of 45° and pure carbon evaporation was at an angle of 90°. Immunogold labeling of freeze-fracture replicas was performed according the protocol described in [29]. Briefly, replicas were thawed in 2.5% SDS in 10 mM Tris and 30 mM sucrose, pH 8.3, and after two changes of the SDS solution replicas were stirred overnight to dissolve organic material not in direct contact to the heavy metal replica sheet.…”

Section: Freeze-fracture Replica Labelingmentioning

confidence: 99%

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Lörinczi

Tsivkovskii

Haase

et al. 2008

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.

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“…; see also for review. The few SDS‐FRL studies on natively cryofixed monolayers relayed on cells cultured on quite exotic substrates such as metal foils and sapphire discs . These ‘substrates’ are also appropriate for resin‐section EM, including postembedding‐IEM , but have, to our knowledge, not been used for cryosection‐IEM (compare in this context).…”

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confidence: 99%

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Schmiedinger

Vogel

Eiter

et al. 2013

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Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non-disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute-long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze-substitution, sample rehydration and cryosection-immunolabelling or with freeze-fracture replica-immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry. In vitro grown vertebrate cells are an indispensable tool for cell biological research, and are widely used for the subcellular localisation of macromolecules at the level of electron microscopy (EM). Yet, the fast and efficient harvest of living, adherent cells without affecting cellular morphology and physiology is still a not satisfactorily solved problem. Thus, the potential of recent improvements (1-3) of cryo-immuno-EM (cryo-IEM) has so far not been fully exploited.To begin with, immunogold-labelling of cryosections according to Tokuyasu (4) has been successfully combined with rapid cryofixation (2,3). The new 'hybrid' approach avoids artefacts resulting from conventional chemical fixation. It is based on cryofixation of native, unfixed specimens, followed by chemical stabilisation of cellular ultrastructure and antigenicity at around −90• C by means of freeze-substitution (FS) and, finally, sample rehydration and postfixation prior to cryosectioning (2,3). So far, this modified Tokuyasu-technique was mainly used for tissues and suspension cultures (2,3,5), but not regularly for adherent cell cultures, with very few exceptions, when relatively large, voluminous cells (HepG2) were cultured on gelatine beads (Cytodex™, Sigma: Ø ∼100 μm; refs. (2,6)). Yet, in our previous studies we found that commercially available beads are less suitable for applying this advanced IEMtechnique to flat and/or small cells, such as mouse embryonic fibroblasts (MEF) with a maximal height of ∼4 μm (ref.(7) and our unpublished data). Considering the adverse ratio of bead-diameter versus cell-height it is evident that most of the area of an average 400 × 400 μm-cryosection is occupied by section profiles of the carrier beads, but not by the cells under investigation. The analysis of a fair amount of cells, however, is mandatory for unbiased stereology (8). Furthermore, sampling of cells grown on beads prior to cryofixation requires time-consuming intermediate enrichment-steps, possibly leading to unwanted physiological and ultrastructural alterations.The second cutting-edge IEM-technique to mention here is sodium dodecyl sulphate-digested freeze-fracture replica labelling (SDS-FRL (1)). So far, SDS-FRL was predominantly applied to chemically fixed tissues e.g. (9); see also (10) for review. The few SDS-FRL studies on natively cryofixed monolayers relayed on...

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A simple and reliable quick-freezing/freeze-fracturing procedure

Cited by 11 publications

References 6 publications

A New Method to Visualize the Helicobacter pylori-associated Lewis^b-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

A New Method to Visualize the Helicobacter pylori-associated Lewis^b-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

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A simple and reliable quick-freezing/freeze-fracturing procedure

Cited by 11 publications

References 6 publications

A New Method to Visualize the Helicobacter pylori-associated Lewisb-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

A New Method to Visualize the Helicobacter pylori-associated Lewisb-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

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A New Method to Visualize the Helicobacter pylori-associated Lewis^b-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling

A New Method to Visualize the Helicobacter pylori-associated Lewis^b-binding Adhesin Utilizing SDS-digested Freeze-fracture Replica Labeling