Conventional freeze-fracture techniques were combined with immunogold labeling and with plastic embedding and sectioning to analyze the distribution of membrane immunoglobulins (mIgs) and their associated intramembrane particles (IMPs) in E-face replicas of murine B-lymphocyte plasma membranes. Immunogold labels were applied to cells after the process of freeze-fracture and replication. Conventional stereoscopic transmission electron microscopic examination of sectioned, labeled replicas (SLRs) revealed that the gold-labeled mIgs were bound to and localized on the outer leaflets of split and replicated membranes. The gold labels were attached to the external determinants of the mIg molecules, which were retained beneath and contiguous with the replicated E-faces. The mIgs were also localized on the external surface of unreplicated microvilli. In addition, thick sections examined by high-voltage transmission electron microscopy (HVEM) revealed large expanses of replica with well-resolved IMPs. mIgs colocalized with small-diameter (less than 60 A) IMPs in E-face replicas of B-lymphocytes whose mIgs were patched by anti-immunoglobulin. Thus, 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.

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, is used in this report.) These post-shadow labelling techniques resemble conventional en bloc labelling techniques except that the labelling reagents must penetrate a thin but discontinuous layer of platinum superimposed on the molecules of interest. In the 'sectioned labelled-replica technique', the replicated and labelled tissues are stained, embedded in plastic and sectioned parallel to the replica-tissue interfaces. In the direct 'labelled-replica techniques', the replicated and labelled samples are freeze-dried or critical point dried, the labelled surfaces are stabilized by carbon coating, and the underlying tissues are dissolved, allowing the labelled-replicas to be examined as conventional freeze-fracture replicas. The unshadowed side of each AChR IMP is shown to retain sufficient biochemical information to permit both immunospeciiic and neurotoxin specific labelling despite formaldehyde fixation, freezing, fracturing, platinum shadowing, and thawing in aqueous media. A new mixed ferricyanide-osmium staining method reveals electron opaque structures spanning the membrane bilayer in the same size, number and distribution as the labelled IMPs. These experiments demonstrate the feasibility of identifying individual IMPs in freeze-fracture replicas and may allow the identification of specific membrane lesions in human disease.

Under conditions commonly used for the preservation of tissue for electron microscopy, substantial amounts of hydrogen ions were produced when either formaldehyde or glutaraldehyde was added to solutions of amines or proteins, or to tissue homogenates. In glycine-aldehyde reactions, hydrogen ions were generated in linear proportion to the glycine concentration over the range of 0.001 M to 0.100 M glycine. In the presence of 1% formaldehyde or 1% glutaraldehyde, 0.3-0.9 equivalents of acid were produced per mole of primary amine. This variation is a function of the specific primary amine used. Acid production increased with increasing formaldehyde concentration but did not iiicrease appreciably with increasing glutaraldehyde concentration. The concentration of amines in tissue is high. These amines are probably responsible for much of the acid generated when aldehydes react with tissue homogenates.In amine-aldehyde reactions, the positive charge originally associated with the parent amine is simultaneously neutralized with the formation of hydrogen ions. Thus, in tissues exposed to aldehydes, there is a decrease in the overall positive charge which is related to the amount of acid generated. The inherent buffering capacity of liver and muscle was not sufficient to prevent large pH decreases when aldehydes were added to homogenates of these tissues. These data suggest that significant but transient pH decreases may occur within cells exposed to aldehydes. To minimize the aldehyde-induced pH decreases, a buf'fer should be chosen so that its pK, is 0.2-0.3 pH units less than the pH desired for a particular experiment. In addition, buffer concentrations of at least 0.10 M are suggested for fixatives in order to provide adequate buffering capacity during tissue preservation.

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Monoclonal antibodies (MAbs) specific for cellobiohydrolase I (CBH I) and endoglucanase I (EG I) were conjugated to 10-and 15-nm colloidal gold particles, respectively. The binding of CBH I and EG I was visualized by utilizing the MAb-colloidal gold probes. The visualization procedure involved immobilization of cellulose microfibrils on copper electron microscopy grids, incubation of the cellulose-coated grids with celiulase(s), binding of MAb-colloidal gold conjugates to cellulase(s), and visualization via transmission electron microscopy. CBH I was seen bound to apparent crystalline cellulose as well as apparent amorphous cellulose. EG I was seen bound extensively to apparent amorphous cellulose with minimal binding to crystalline cellulose.