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Diagnostic immunoelectron microscopy in surgical pathology: assessment of various tissue fixation and processing
S L Mount1, D J Taatjes, M von Turkovich
1Department of Pathology, University of Vermont, Burlington 05405.
Insights
Investigating tissue processing for diagnostic pathology, this study shows various methods successfully localize chromogranin A using immunoelectron microscopy. Archival and snap-frozen tissues can be used, with Lowicryl K4M embedding showing promise.
Area of Science:
- Pathology
- Microscopy
- Immunohistochemistry
Background:
- Immunoelectron microscopy (IEM) is crucial for ultrastructural diagnosis.
- Standard fixation and embedding protocols may limit IEM applications in surgical pathology.
Purpose of the Study:
- To evaluate diverse tissue processing methods for IEM.
- To assess the feasibility of using archival or snap-frozen tissues for IEM.
Main Methods:
- Tested seven fixation and embedding protocols on normal human small bowel mucosa.
- Localized chromogranin A using protein A-gold labeling.
- Examined archival neuroendocrine tumor tissues processed via various methods, including formalin-fixed, snap-frozen, and Lowicryl K4M embedding.
Main Results:
- Precise ultrastructural localization of chromogranin A in dense core granules was achieved across all tested methods.
- Successful retrieval and processing of previously formalin-fixed or snap-frozen tissues were demonstrated.
- Protein A-gold labeling effectively localized chromogranin A in all sample types.
Conclusions:
- Tissue processing without prior IEM planning can yield successful results.
- Lowicryl K4M embedding preserves antigenicity and is a superior resin for diagnostic IEM.
- Expanded use of IEM in surgical pathology is feasible with optimized protocols.
Abstract:
We have investigated various tissue fixation and embedding protocols in an effort to allow expanded use of immunoelectron microscopy in diagnostic surgical pathology. A sample of normal human small bowel mucosa was processed using seven different methods for subsequent postembedding localization of chromogranin A. In addition, several archival cases of neuroendocrine tumors previously fixed and routinely embedded for electron microscopy, stored in formalin, or snap-frozen were retrieved and variously processed for chromogranin A localization at the ultrastructural level. Precise localization of chromogranin A in dense core granules was achieved with protein A-gold on sections from all of the processing methods. The methods included retrieval into mild fixative of previously formalin-fixed or snap-frozen tissues followed by embedding in Lowicryl K4M (Polysciences Ltd., Eppelheim, Germany). Thus, tissue processed without foresight of the need for immunoelectron microscopic localization can be successfully used. Since embedding of tissues in Lowicryl K4M has been shown to preserve a variety of antigens, it may prove to be a superior resin for use in diagnostic immunoelectron microscopy.
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