Freeze‐fracture cytochemistry: Review of methods

Abstract

"Freeze-fracture cytochemistry" encompasses a diversity of recently developed techniques in which freeze-fracture and cytochemistry are combined. Cytochemical labeling may, in principle, be integrated into one of three basic points in the standard freeze-fracture procedure; 1) before the specimen is frozen, 2) after it has been fractured, or 3) after it has been platinum shadowed and/or carbon coated. Visualization of the labeled cellular structures can be achieved by a variety of different methodologies. For example, the markers (usually colloidal gold particles) may be viewed embedded within a replica, or attached to it via fragments of membrane (or other cellular components). Sectioning is a central strategy in a number of techniques, either in combination with or in place of replication. The different combinations of methods that have been devised are not, for the most part, alternative ways of arriving at the same result; each provides quite distinct information about specific classes of membrane component or other structure in the cell. The purpose of this review is to present, within a single article, a systematic survey of the full range of techniques currently available in freeze-fracture cytochemistry. Emphasis is placed on explaining the principles underlying the methods and on illustrating their applications. With the success recently achieved, freeze-fracture cytochemistry has moved from the phase of experimental development to a position in which it may be expected increasingly to make significant contributions across a wide spectrum of problems in cell and membrane biology.

Topographical distribution of phospholipids in boar sperm plasma and intracellular membranes as revealed by freeze-fracture cytochemistry.

“…The other consists of the preparation of thin sections of fracturelabeled specimens. The important feature of fracture-label is its ability to expose individual membrane leaflets of the plasma and intracellular membranes as well as other cytoplasmic components and to render them directly accessible to the labeling probes (for reviews see Severs, 1989Severs, ,1995. The label-fracture technique is useful for studying surface domains in highly regionalized cells such as the spermatozoa presented in this study.…”

Co-localization of Dystrophin and β-Dystroglycan Demonstrated in En Face View by Double Immunogold Labeling of Freeze-fractured Skeletal Muscle

“…To achieve this, we applied the fracture-label technique, which permits high-resolution visualization of labeled membrane components both in sections and in platinumcarbon replicas of the fractured tissue face (Pinto da Silva et al 1986;Severs 1995). Investigations involving the double labeling of dystrophin and ␤ -dystroglycan have been made by Wakayama's group using either mouse (Wakayama et al 1995) or human muscle .…”

“…Freeze-fracturing in this way generates fracture planes through the sample that preferentially follow the membrane planes, as in conventional freeze-fracture electron microscopy (Pinto da Silva et al 1981aSilva et al ,b,1986Severs 1995). The crushed (fractured) specimens were then allowed to thaw in the presence of pre-cooled fixative in 30% glycerol.…”

“…The overall distribution of labeling sites was reasonably even, with no localized concentrations of immunogold. There were some unlabeled areas on the P half-leaflet that could be caused by loss of gold during cleaning of the replica or reorganization of the membrane during thawing (Severs 1995). Sporadic clumping of the gold particles was attributed to multiple binding to the primary antibody by the secondary conjugate (Figure 2A).…”

Biodegradable polymersomes for drug delivery : circulation kinetics and biodistribution, modulated drug delivery and cellular uptake

“…2.7b) [18]. Freeze-fracture TEM can be used to study the internal structure of Ps by fracturing and etching the frozen samples [136,137]. However, electrons cannot deeply penetrate into membrane of Ps and the quality of the photographs is dependent on the optical properties of the polymers applied [138][139][140][141].…”