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Enhanced immunohistochemical resolution of claudin proteins in glycolmethacrylate-embedded tissue biopsies
Jane E Collins1, Adam Kirk, Sara K Campbell
1Division of Infection, Inflammation, and Immunity, University of Southampton Medical School, Southampton, UK. jec3@soton.ac.uk
Insights
Glycol methacrylate (GMA) embedding improves immunohistochemistry for claudin proteins by preserving epitopes and enabling thinner sections. This method enhances antigen accessibility and maximizes data from small tissue biopsies.
Area of Science:
- Biochemistry
- Histology
- Immunohistochemistry
Background:
- Conventional tissue processing for immunohistochemistry, such as formaldehyde fixation and wax embedding, often hinders antigen accessibility and antibody binding.
- Harsh antigen retrieval methods are frequently required, potentially disrupting antibody localization patterns.
- Cryopreservation or wax embedding necessitates thick tissue sections (>6 μm), limiting serial section analysis.
Purpose of the Study:
- To evaluate glycol methacrylate (GMA) tissue embedding as an alternative method for improved immunohistochemistry.
- To assess the compatibility of GMA with epitope preservation for antibody reagents.
- To enhance the analysis of claudin proteins and other antigens in small tissue biopsies.
Main Methods:
- Tissue fixation in acetone followed by embedding in glycol methacrylate (GMA), a water-miscible resin.
- Sectioning of GMA-embedded tissue to produce thin sections (2 μm).
- Immunohistochemical localization of claudin proteins and other antigens using antibody reagents.
Main Results:
- GMA embedding preserves epitopes that are often destroyed by chemical cross-linking fixatives.
- The method maintains tissue hydration, reducing shrinkage and improving morphology.
- Thin sequential sections (2 μm) facilitate the analysis of multiple antigens and maximize information from small biopsies.
Conclusions:
- Glycol methacrylate (GMA) tissue embedding offers significant advantages over conventional methods for immunohistochemistry.
- This technique enhances antigen accessibility and preserves epitope integrity, leading to more accurate protein localization.
- GMA embedding is particularly beneficial for analyzing small human tissue biopsies, maximizing diagnostic yield.
Abstract:
There are a number of disadvantages with conventional tissue immunohistochemistry for accurate -localisation of claudin proteins. Traditionally, tissue cryopreservation or formaldehyde fixation with wax embedding is utilised prior to sectioning and antibody localisation. Wax embedding gives better morphological preservation than frozen tissue, but the required use of chemical cross-linking fixatives renders many antigens inaccessible to antibody binding or results in subsequent disruption of antibody localisation patterns due to the use of harsh antigen retrieval methods. Use of frozen or wax-embedded tissue also requires the cutting of relatively thick>6-μm sections, making the interrogation of serial sections very limited. The use of glycolmethacrylate (GMA) tissue embedding with fixation in acetone is compatible with epitope preservation for many antibody reagents that are often destroyed by chemical cross-linking fixatives. GMA is a water-miscible embedding resin that maintains tissue hydration during processing, thus reducing tissue shrinkage, while embedding and cutting in the polymerised resin physically supports the tissue, thus improving morphology. This method also facilitates the cutting of 2-μm sequential sections for analysis of multiple antigens and maximises the information available from small tissue biopsies from human clinical sources.
