Human neutrophil granule heterogeneity: immunolocalization studies using cryofixed, dried and embedded specimens

The heterogeneity in human neutrophil granules was examined by the ultrastructural localization of a series of antigens which have been previously identified with neutrophil granules by either physical separation or biochemical/biological techniques. All samples were prepared by cryofixation and molecular distillation drying (LifeCell Process), a two-step physical method that achieves cryofixation by metal mirror freezing and drying by the controlled, incremental heating of cryofixed samples in an ultrahigh vacuum. After drying, the samples were either exposed to vapor-phase osmium followed by embedment in Spurr resin, or they were exposed to formaldehyde vapor followed by embedment in Araldite resin. An indirect streptavidincolloidal gold procedure was used for immunoelectron microscopy on ultrathin sections. Subcellular ultrastructural morphology of neutrophils prepared by this method was good compared to standard electron microscopic techniques and superior compared to comparable, published electron microscopic cryomethods applied to neutrophils. Immunogold localization of myeloperoxidase, cathepsin G, lysozyme, lactoferrin, beta 2-microglobulin, and CD-15 antigens showed high intensity and specificity of labeling in the intracellular granules. Patterns of labeling varied from antigen to antigen, demonstrating granule heterogeneity both within and among neutrophils. This methodology is useful in the exploration and definition of granule heterogeneity and function.