Labelled‐replica techniques: post‐shadow labelling of intramembrane particles in freeze‐fracture replicas

  title={Labelled‐replica techniques: post‐shadow labelling of intramembrane particles in freeze‐fracture replicas},
  author={John E Rash and Timothy J. A. Johnson and C. Sue Hudson and F D Giddings and W F Graham and Mohyee E. Eldefrawi},
  journal={Journal of Microscopy},
Three methods are described for direct post‐fracture, post‐shadow labelling of individual classes of intramembrane particles (IMPs) in freeze‐fracture replicas of biological membranes. The P‐face IMPs corresponding to the acetylcholine receptor complexes (AChRs) of vertebrate neuroeffector junctions are identified by post‐replication labelling with ferritin‐antibody complexes and with neurotoxin‐biotin‐avidin‐colloidal gold affinity ligands. (The freeze‐etch nomenclature of Branton et al., 1975… 

Composite replicas: methodologies for direct evaluation of the relationship between intramembrane and extramembrane structures.

While some unlabelled or lightly labelled surface elements can be lost from fractured membranes, ligands stabilize elements and reduce their loss apparently by cross-linking them.

Postreplication labeling of E-leaflet molecules: membrane immunoglobulins localized in sectioned, labeled replicas examined by TEM and HVEM.

Postreplication E-surface labeling of split and replicated membranes is a high-resolution technique that is suitable for the study of membrane protein distribution in E-face replicas and contiguous nonreplicated tissue.

Freeze‐fracture cytochemistry: a simplified guide and update on developments

Recent developments have centred on the use of replicas to stabilize half‐membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen— specifically, turning it over during processing—so that additional structural information can be obtained.

Label-fracture: a method for high resolution labeling of cell surfaces

"Label-fracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-f Fracture morphology of exoplasmic fracture faces.

Freeze-fracture cytochemistry: review of methods.

  • N. Severs
  • Biology
    Journal of electron microscopy technique
  • 1989
The purpose of this review is to present a systematic survey of the full range of techniques currently available in freeze-fracture cytochemistry, with emphasis on explaining the principles underlying the methods and on illustrating their applications.

Freeze—fracture and freeze—etch electron microscopy of membrane proteins

Using freeze—fracturing, investigators have visualized a broad spectrum of membrane morphologies that have served to refine and change earlier concepts of membrane structure, particularly with respect to the organization of membrane proteins.

(Na ÷ + K÷)-ATPase Correlated of Intramembrane Particles in of Cultured Chick Myotubes with a Major Group Freeze-Fracture Replicas

It is concluded that the intramembrane particles patched by double antibody cross-linkage of the ( Na, K)-ATPase are caused by (Na, K) ATPase molecules in the fracture plane.



Freeze-fracture cytochemistry: replicas of critical point-dried cells and tissues after fracture-label.

This method allows direct, high-resolution, two-dimensional chemical and immunological characterization of the cellular membranes in situ, as well as detection of sites within cross-fractured cytoplasm and extracellular matrix.

Fracture-label:O cytochemistry of freeze-fracture faces in the erythrocyte membrane.

Fracture-label can be applied to the cytochemical characterization of the cellular components exposed by freeze-fracture of isolated cells and tissues.


A modification of the freeze-fracturing technique to permit observation of replicas of both sides of the fracture is described. It has been used to study mouse liver cell membrane structure.


Results provide further support for the idea that membrane-bound glycoproteins are oriented so that their carbohydrate-rich segments, which bear the antigenic sites and receptors, are exposed to the external medium, while hydrophobic segments of the same molecules interact with lipids, and possibly other proteins, to form the intramembranous particles.

Prefracture and cold-fracture images of yeast plasma membranes

These findings demonstrate that the prefracture image results from the fracturing of specimens at some temperature above 230 degrees K, no t from fracturing specimens atSome temperature between 173 degrees and 77 degrees K and not from the use of "starved" yeast cells.

Appearance and distribution of surface proteins of the human erythrocyte membrane. An electron microscope and immunochemical labeling study

It is suggested that a close dynamic association may exist between the integral and peripheral protein components of the membrane, such that immobilization of one component can restrict the lateral mobility of others.

STUDIES OF EXCITABLE MEMBRANES: I. Macromolecular Specializations of the Neuromuscular Junction and the Nonjunctional Sarcolemma

Evidence is assembled which indicates that these large transmembrane macromolecules may represent the morphological correlate for functional acetylcholine receptor activity in mammalian motor endplates.

Freeze-fracture autoradiography: feasibility

We have shown that the combination of freeze-fracture with electron microscope autoradiography can be developed into a technique for correlating the molecular structure of the biological membrane

Freeze fracture studies of human neuromuscular junctions. Membrane alterations observed in myasthenia gravis.

The destructive alterations observed in junctional fold membranes in myasthenia gravis endplates are correlated with decreased acetylcholine sensitivity and to removal of endplate acetylCholine receptors by immunologic processes.