CRYOFIXATION COMBINED WITH PHYSICAL DEHYDRATION FOR QUANTITATIVE IMMUNOELECTRON CYTOCHEMISTRY

Common methods for preparing samples for immunoelectron microscopy inv olve 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 techniq ues have been evaluated with regard to the sizes of epoxy resin embedd ed rat somatotrophic secretory granules as well as the immunolabeling densities over these granules. The measurements were performed by comp uterized image analysis using electron microscopy in transmission (TEM ) and scanning transmission (STEM) modes, which allowed us to define t he immunolabeling in detail. The embedded secretory granules showed th e same diameters after GA (2 hr) with CD and GA (15 min) with CF and F S, but were smaller after CF-FS, and smallest after GA (15 min) with C F and FD. The highest labeling density appeared after GA (15 min) and physical dehydration, in particular after freeze substitution. Based o n our STEM pictures a new factor for evaluating and interpreting immun olabeling of granules is introduced; the ''accessible immunogold label ing surface.'' It defines the fraction of the epoxy resin surface that is labeled and varies with the preparation methods. By using this fac tor, an order of labeling densities/mu m(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 c ombination of a large accessible area and accurate preservation of the antigenicity of the hormones in the granules.