Immunoelectron microscopy of frozen hydrated blood platelets

Abstract

We have previously shown that ultrarapid freezing followed by cryomicroscopy provides a uniquely effective preservation of platelet ultrastructure, particularly in its cytoskeleton and membrane systems. However, a limitation of cryomicroscopy of frozen hydrated specimens is the lack of information about the localization of specific proteins within the cell. Methods for immunolocalization of antigens on material that has been rapidly frozen, freeze-substituted and embedded in immunocompatible resins have excited considerable interest among biologists, because of the high quality of preservation afforded by fast-freezing techniques. However, the extent to which fixation and dehydration affect the distribution of antigens is not known. Here we describe a method for imaging immunolabeled cells in the frozen hydrated state, avoiding the destructive steps of dehydration.Blood platelets were isolated from citrated whole blood by differential centrifugation and washed in Hank’s balanced salts. Washed platelet suspensions were placed on formvar coated, carbon stabilized gold finder grids and placed in a humid chamber at 37°C for 30 minutes to allow adhesion and spreading of the cells.