Cryofixation combined with physical dehydration for quantitative immunoelectron cytochemistry.

Abstract

Common methods for preparing samples for immunoelectron microscopy involve glutaraldehyde fixation (GA) followed by chemical dehydration (CD) or cryofixation (CF) succeeded by physical dehydration, i.e., freeze drying (FD) or freeze substitution (FS). The effects of these techniques have been evaluated with regard to the sizes of epoxy resin embedded rat somatotrophic secretory granules as well as the immunolabeling densities over these granules. The measurements were performed by computerized image analysis using electron microscopy in transmission (TEM) and scanning transmission (STEM) modes, which allowed us to define the immunolabeling in detail. The embedded secretory granules showed the same diameters after GA (2 hr) with CD and GA (15 min) with CF and FS, but were smaller after CF-FS, and smallest after GA (15 min) with CF and FD. The highest labeling density appeared after GA (15 min) and physical dehydration, in particular after freeze substitution. Based on our STEM pictures a new factor for evaluating and interpreting immunolabeling of granules is introduced; the "accessible immunogold labeling surface." It defines the fraction of the epoxy resin surface that is labeled and varies with the preparation methods. By using this factor, an order of labeling densities/micron 2 over the accessible areas could be established for the different techniques: GA-CF-FS > CF-FS > GA-CF-FD > GA-CD. The high labeling after GA-CF-FS may be due to the combination of a large accessible area and accurate preservation of the antigenicity of the hormones in the granules.