Enhanced Immunohistochemical Resolution of Claudin Proteins in Glycolmethacrylate-Embedded Tissue Biopsies

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.