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

Publisher Website
Google Scholar
Abstract
Three methods are described for direct postfracture, postshadow labeling of individual classes of intramembrane particles (IMP) in freeze-fracture replicas of biological membranes. The P-face IMP corresponding to the acetylcholine receptor complexes (AChR) of vertebrate pseuroeffector juntions are identified by postreplication labeling with ferritin-antibody complexes and with neurotoxin-biotin-avidin-colloidal Au affinity ligands. (The freeze-etch nomenclature of Branton et. al., 1975, is used in this report.) These postshadow labeling techniques resemble conventional en bloc labeling techniques except that the labeling reagents must penetrate a thin but discontinuous layer of Pt superimposed on the molecules of interest. In the sectioned labeled-replica technique, the replicated and labeled tissues [rat muscle and Torpedo electroplax vesciole] are stained, embedded in plastic and sectioned parallel to the replica-tissue interfaces. In the direct labeled-replica techniques, the replicated and labeled samples are freeze-dried or critical point dried, the labeled surfaces are stabilized by C coating, and the underlying tissues are dissolved, allowing the labeled-replicas to be examined as conventional freeze-fracture replicas. The unshadowed side of each AChR IMP retains, sufficient biochemical information to permit both immunospecific and neurotoxin specific labeling despite formaldehyde fixation, freezing, fracturing, Pt shadowing, and thawing in aqueous media. A new mixed ferricyanide-osmium staining method reveals electron opaque structures spanning the membrane bilaryer in the same size, number and distribution as the labeled IMP. The feasibility of identifying individual IMP in freeze-fracture replicase was demonstrated; this may allow the identification of specific membrane lesions in human disease.

This publication has 50 references indexed in Scilit: