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Updated: Aug 15, 2026
Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections
Published on: November 14, 2016
Cryosubstitution dehydration of aldehyde-fixed tissue: a favorable approach to quantitative immunocytochemistry
Insights
Cryosubstitution (CS) dehydration followed by Lowicryl HM20 embedding is optimal for quantitative immunoelectron microscopy (IEM). This method preserves cellular structure, ensures uniform immunolabeling, and enables non-interfering double labeling.
Area of Science:
- Electron Microscopy
- Cell Biology
- Biochemistry
Background:
- Quantitative immunoelectron microscopy (IEM) requires tissue processing methods that maintain cellular integrity and antigen accessibility.
- Evaluating different tissue preparation techniques is crucial for optimizing IEM results.
Purpose of the Study:
- To assess various tissue-processing procedures for quantitative immunoelectron microscopy (IEM).
- To identify methods that minimize cellular shrinkage, ensure uniform immunolabeling efficiency, and allow for non-interfering double labeling.
Main Methods:
- Comparison of tissue processing techniques including hydration, polyacrylamide embedding, and dehydration via cryosubstitution (CS) or progressive lowering of temperature (PLT).
- Embedding in resins like Lowicryl HM20, LR Gold, or LR White.
- Evaluation using a gelatin model system and rat pancreatic tissue.
Main Results:
- Cryosubstitution (CS) dehydration followed by embedding at temperatures below -45°C using Lowicryl HM20 yielded satisfactory results in all investigated aspects.
- This CS-HM20 procedure prevented shrinkage in aldehyde-fixed material, unlike other non-aqueous embedding methods.
- Homogeneous labeling efficiency was achieved by equalizing antigen accessibility, comparable to the polyacrylamide method.
- CS-HM20 sections allowed for successful double labeling without interference and presented well-defined ultrastructure.
Conclusions:
- The CS dehydration and Lowicryl HM20 embedding method is highly suitable for quantitative IEM.
- This technique offers advantages in preserving ultrastructure, achieving uniform labeling, and enabling multiplexed detection.
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 degrees C or progressive lowering of the temperature (PLT). We found that only CS dehydration followed by embedding at temperatures below -45 degrees 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.
