Conventional freeze-fracture replicas provide for high resolution,
three-dimensional replication of the internal macromolecular architecture of
cell membranes, but they have the distinct disadvantage that no biologically
rele¬vant macromolecules remain for subsequent in situ labelling and
positive identification. Recently, several ingenious freeze-fracture
techniques have been devised to localize biochemical components before the
tissues are diges-ted away. These methods include (a) freeze-fracture
autoradiography of the frozen whole mount preparation (1, 2) and (b) deep
etching (freeze-drying) to expose immunoglobins or other large protein
markers attached to membrane surfaces (3). However, freeze-fracture
autoradiography has insufficient resolution to permit localization of labels
to individual intramembrane particles (IMPs) exposed by the cleaving
process, while deep etching to reveal membrane surfaces does not reveal
IMPs. To overcome some of these difficulties, we recently introduced the
"sectioned replica technique" to provide a method for direct and unambiguous
correlation of freeze-fracture and thin section images within a single
electron micrograph (4). The fractured and replicated tissues are thawed,
but instead of digesting the tissues, the replica-tissue sandwich is
post-fixed in 30% buffered glycerol solution con-taining 1%
OsO4, post-stained, embedded in plastic, and
sectioned parallel to the replica-tissue interface. Using this
sectioned-replica technique, we have been able to obtain (a) pre-fracture
radioisotope-labelled mouse neuromuscular junctions and (b) pre- and
post-fracture immunoferritin and colloidal gold-labelled membrane fragments
and intact neuromuscular junctions.
Cross-linking of erythrocyte membrane proteins by periodate and intramembrane particle distribution