Optimal preparatory procedures of cryofixation for immunocytochemistry

To examine the optimal preparatory procedures of cryofixation for immunocytochemistry, the labeling density over the antigenic sites in cells processed by various protocols of freeze-substitution and embedding was quantitatively evaluated. Fresh tissue blocks of gerbil parotid gland were quickly frozen by a metal contact method using liquid helium and freeze-substituted with one of the following media: 4% OsO4 in acetone or 0.4% OsO4 in acetone or 0.3% glutaraldehyde in acetone. They were then embedded in either an Epon-Araldite mixture or Araldite 6005, which were polymerized at 60 degrees C and 50 degrees C, respectively. Some frozen samples substituted with aldehyde-containing acetone were embedded in Lowicryl K4M (polymerized at -30 degrees C). Immunocytochemical localization of amylase was examined by indirect immunostaining by using antigerbil parotid amylase antibody and protein A/gold complex. Thin sections of epoxyresin-embedded materials were treated with oxidizing agents before immunostaining. The central dense core of heterogeneous secretory granules in the acinar cells was heavily labeled with immunogold, regardless of substitution media and embedding resins employed. The labeling density on thin sections of all the cryofixed materials examined was about 1.5 times or more as high as in those processed by conventional chemical fixation. The highest value of the labeling density was obtained from material which was substituted with 0.3% glutaraldehyde in acetone and embedded in Araldite 6005. Substitution with osmium-containing acetone appeared not to seriously affect immunoreactivity of the antigenic sites and was advantageous because of the distinctive images of membranes. Advantages and disadvantages of the individual protocols employed are discussed.