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Tissue preparation for histochemistry: fixation, embedding, and antigen retrieval for light microscopy
Tim D Hewitson1, Belinda Wigg, Gavin J Becker
1Department of Nephrology, The Royal Melbourne Hospital, Melbourne, VIC, Australia.
Insights
Histochemistry uses specialized assays on tissue samples for in situ biological study. Proper tissue preparation, including freezing or paraffin embedding, is crucial for accurate results and antigen preservation.
Area of Science:
- Histochemistry
- Immunohistochemistry
- Molecular Biology Assays
Background:
- Histochemical techniques enable in situ study of tissue and organ biology.
- Success relies heavily on appropriate tissue preparation methods.
Purpose of the Study:
- To outline essential tissue preparation steps for routine histochemical analysis.
- To compare frozen and paraffin-embedded tissue preparation for histological analysis.
Main Methods:
- Describing basic tissue preparation for histochemical analysis.
- Discussing frozen tissue preparation for minimal epitope alteration.
- Detailing paraffin embedding for enhanced structural integrity.
Main Results:
- Frozen tissue may offer superior staining due to less epitope alteration.
- Paraffin embedding provides better morphology but can reduce antigenicity.
- Antigen retrieval techniques can mitigate antigen loss in paraffin-embedded tissues.
Conclusions:
- Adequate tissue preparation is fundamental for successful histochemical analysis.
- Choosing between frozen and paraffin-embedded methods depends on balancing morphological detail and antigen preservation.
Abstract:
A number of techniques have been developed to use chemical, immunological, and molecular biology assays in histological material. Collectively termed histochemistry, these techniques have allowed us to better understand tissue and organ biology in situ. Success with each of these methods is dependent on the adequate preparation of material. In this article, we describe the basic steps required to prepare tissue for routine histochemical analysis.Histochemical techniques routinely use frozen and paraffin-embedded tissue as a basis for cellular and morphological analysis. Freezing tissue results in less alteration to epitopes and therefore may offer improved staining characteristics compared to techniques based on paraffin embedding. As in conventional histology, the use of fixation and embedding in more rigid media such as wax offers a number of potential advantages related to improved structural detail. Improvements in morphology may however be offset by a loss of antigens. The careful application of antigen retrieval techniques may overcome these deficiencies.
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01:03Fixation and Sectioning
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01:20Preparation of Samples for Electron Microscopy