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Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique.

Robenek H, Severs NJ - Biol Proced Online (2008)

Bottom Line: Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy.Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure.Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.

View Article: PubMed Central - PubMed

Affiliation: University of Münster, Domagkstr. 3D-48149 Münster, Germany.

ABSTRACT
Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried, the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique, samples are frozen, fractured and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.

No MeSH data available.


Related in: MedlinePlus

Fig. 11                  Distribution of butyrophilin in concavely fractured milk fat globules. Intensive labeling of butyrophilin occurs on both (A) the P face (PF) of the monolayer and (B) E face (EF) of the bilayer. Butyrophilin labeling on the bilayer E face occurs exclusively in a network of more or less prominent ridges. Bars: 0.2 µm
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f11: Fig. 11 Distribution of butyrophilin in concavely fractured milk fat globules. Intensive labeling of butyrophilin occurs on both (A) the P face (PF) of the monolayer and (B) E face (EF) of the bilayer. Butyrophilin labeling on the bilayer E face occurs exclusively in a network of more or less prominent ridges. Bars: 0.2 µm

Mentions: Furthermore, adipophilin is localized in the bilayer surrounding the secreted milk fat globule (Fig. 9B) and in the monolayer enclosing the lipid droplet (Fig. 10). Xanthine oxidoreductase is diffusely distributed in the lipid droplet monolayer (Fig. 10). Intensive labeling of butyrophilin occurs on both the P face of the monolayer and the E face of the bilayer (Fig. 11, 12). Importantly, butyrophilin labeling on the bilayer E face occurs as a network of ridges that tightly appose and match the distribution of butyrophilin label in the monolayer (Fig. 12A). Double labeling shows that the distribution of butyrophilin differs from that of adipophilin. Whereas butyrophilin labeling occurs both on the P face of the monolayer and on the E face of the bilayer, adipophilin is labeled on the P face of the monolayer, but not on the E face of the bilayer (Fig. 12B). The findings from FRIL suggest that while adipophilin-rich domains in the plasma membrane may be linked to secretory granule positioning at the cell surface, butyrophilin-butyrophilin interactions between monolayer and bilayer are responsible for envelopment of the granule by the plasma membrane and its release from the cell (26).


Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique.

Robenek H, Severs NJ - Biol Proced Online (2008)

Fig. 11                  Distribution of butyrophilin in concavely fractured milk fat globules. Intensive labeling of butyrophilin occurs on both (A) the P face (PF) of the monolayer and (B) E face (EF) of the bilayer. Butyrophilin labeling on the bilayer E face occurs exclusively in a network of more or less prominent ridges. Bars: 0.2 µm
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2275045&req=5

f11: Fig. 11 Distribution of butyrophilin in concavely fractured milk fat globules. Intensive labeling of butyrophilin occurs on both (A) the P face (PF) of the monolayer and (B) E face (EF) of the bilayer. Butyrophilin labeling on the bilayer E face occurs exclusively in a network of more or less prominent ridges. Bars: 0.2 µm
Mentions: Furthermore, adipophilin is localized in the bilayer surrounding the secreted milk fat globule (Fig. 9B) and in the monolayer enclosing the lipid droplet (Fig. 10). Xanthine oxidoreductase is diffusely distributed in the lipid droplet monolayer (Fig. 10). Intensive labeling of butyrophilin occurs on both the P face of the monolayer and the E face of the bilayer (Fig. 11, 12). Importantly, butyrophilin labeling on the bilayer E face occurs as a network of ridges that tightly appose and match the distribution of butyrophilin label in the monolayer (Fig. 12A). Double labeling shows that the distribution of butyrophilin differs from that of adipophilin. Whereas butyrophilin labeling occurs both on the P face of the monolayer and on the E face of the bilayer, adipophilin is labeled on the P face of the monolayer, but not on the E face of the bilayer (Fig. 12B). The findings from FRIL suggest that while adipophilin-rich domains in the plasma membrane may be linked to secretory granule positioning at the cell surface, butyrophilin-butyrophilin interactions between monolayer and bilayer are responsible for envelopment of the granule by the plasma membrane and its release from the cell (26).

Bottom Line: Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy.Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure.Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.

View Article: PubMed Central - PubMed

Affiliation: University of Münster, Domagkstr. 3D-48149 Münster, Germany.

ABSTRACT
Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried, the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique, samples are frozen, fractured and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.

No MeSH data available.


Related in: MedlinePlus