Plastic Embedding Techniques for Light Microscopy Histological Studies

This thesis collates, records and reviews pioneering studies performed by the author on the formulation, use, advantages, and applications of acrylic plastic embedding media for the histological examination of tissue using light microscopy, with particular emphasis on histochemical and molecular techniques. Nine key papers by the author are reviewed. The first relates to a number of modifications of an established method for the histological examination and investigation of metabolic bone diseases. As a result it was reported that the procedure had resulted in significant time saving without detrimentally affecting quality, and has been used for investigating approximately 10,000 cases of metabolic bone diseases for diagnosis. In a further separate development, the enzymes lactase and sucrase were first reported in plastic-embedded jejunum. Subsequently it was shown that these two important disaccharidases for assessing malabsorption were sensitive to specific processing agents and could easily be lost, resulting in false negative staining. However, by using a specific processing schedule and times, the author was able to demonstrate how best tissue could be processed to retain these and other enzymes in various plastic embedding media. A number of procedures were initiated for the application of IHC on plastic- embedded tissue that included the development of a modified plastic with new preparatory techniques, and changes in immunocytochemical staining protocols to enable IHC to be routinely performed. Previously, this had been regarded as technically not possible. Later in a series of papers published, procedures were described where the use and evaluation of enzyme, and/or antigen retrieval techniques using microwave heating and/or pressure cooking were assessed. Numerous antigens were successfully demonstrated, and subsequently specific diagnostic applications on various tissues were reported, including extensive routine use on approximately 35,000 cases to date of bone marrow trephines. In a further development, the application of ISH techniques for the detection of mRNA in chick tissue for the demonstration of transcription Sox genes 11 and 21 was reported, which subsequently was combined with IHC for bromodeoxyuridine or neurofilament protein for simultaneous double staining on the same section.