Cryosubstitution dehydration of aldehyde-fixed tissue: A favorable approach to quantitative immunocytochemistry

Abstract

Several tissue-processing procedures were studied for their applicability in quantitative immunoelectron microscopy (IEM). Three aspects were mainly considered: maintenance of the natural dimensions of cellular structures (no shrinkage), equal efficiency of immunolabeling throughout a specimen, and the possibility of non-interfering double labeling. These aspects were studied in a gelatin model system and in rat pancreatic tissue, which we subjected to different processing procedures. Some aldehyde-fixed specimens were kept hydrated and prepared for cryosectioning directly or after embedding in polyacrylamide (PAA). Other samples were dehydrated and embedded in different resins, i.e., Lowicryl HM20, LR Gold, or LR White. Dehydration was performed under conditions of cryosubstitution (CS) at -90°C or progressive lowering of the temperature (PLT). We found that only CS dehydration followed by embedding at temperatures below -45°C, which is compatible with Lowicryl HM20, gave satisfactory results in all three aspects investigated. We have previously introduced this procedure for IEM of glycolipids. Unlike in other non-aqueous embedding procedures, aldehyde-fixed material can be embedded via this CS-HM20 procedure without detectable shrinkage. The method also provides homogeneous labeling efficiency by equalizing the accessibility of antigens, irrespective of the original matrix in which they are packed. In this respect the CS-HM20 method equals the previously introduced but more bothersome PAA method. In addition, two-sided labeling of CS-HM20 sections allows double labeling without mutual hindrance of both immunoreactions, and these sections present a well-defined ultrastructure.