A new histological embedding method by low-temperature polymerisation of methyl methacrylate allowing immuno- and enzyme histochemical studies on semi-thin sections of undecalcified bone marrow biopsies

Abstract

A new procedure of embedding in methyl methacrylate (MMA) is introduced, which enables immunostaining by preservation of cellular epitopes. This could be achieved by reduction of polymerisation temperature from ca. 60 degrees C to 22 degrees C within the core of tissue blocks. Reduction of the polymerisation temperature is due to destabilisation of acrylate monomer, reduction of catalyst, exclusion of molecular oxygen, chemical initiation and reduction of environmental temperature. This results in good preservation of antigens and enzymes in the haematopoietic and lymphatic tissue of bone marrow as well as lymphoid, epithelial and mesenchymal markers in other tissues, comparable to paraffin embedding. Results are demonstrated by application of monoclonal and polyclonal antibodies and by demonstration of enzyme activity conventionally used in haematology.

Immunohistochemistry of matrix markers in Technovit 9100 New®-embedded undecalcified bone sections

“…Polymerisation of MMA at 19-22°C yielded enzymatic preservation of chloracetate esterase, elastase, lysozyme, factor VIII-related-antigen, GPIIIA, CD45, MB1, MT1, Ki-67 and others (Bernhards et al, 1992;Georgii et al, 1995;Lebeau et al, 1995). Even polymerisation of MMA at 40°C yielded immunolabelling of human collagen type I, III, IV, fibronectin, and antilaminin (Lucena et al, 1997).…”

Pre-osteoblastic Proliferation Assessed with BrdU in Undecalcified, Epon-embedded Adult Rat Trabecular Bone

“…Glycol-methacrylate (GMA) has been shown to provide excellent results for immunohistochemistry of non-collagenous matrix proteins in bone tissue (Groot et al 1986;Ingram et al 1993) and MMA for bone marrow antigens (Bernhards et al 1992). However, as emphasized by Xiang and Markel (1995), our experiments confirmed that MMA was not a suitable embedding medium for BrdU labeling.…”

Tyramide Signal Amplification: An Enhanced Method for Immunohistochemistry on Methyl-Methacrylate-Embedded Bone Marrow Trephine Sections

“…Despite the excellent preservation of cellular morphology by the methyl methacrylate embedding of bone marrow trephines [2, 3, 5], additional immunohistochemical investigations are required for the classification of leukemia and lymphoma as well as the determination of residual disease. However, limitations concerning the immunohistochemical staining of trephines may be due to different times of fixation or tissue processing resulting in a loss of antigenicity.…”

“…However, despite the excellent preservation of morphology in methyl-methacrylate-embedded bone marrow trephines, an additional immunohistochemical evaluation of bone marrow trephine biopsies is required for the classification of leukemia and lymphomas as well as for the monitoring of residual disease. In the initially developed techniques of methyl methacrylate embedding of bone marrow trephines [2,3,4], immunohistochemistry could not be established due to extremely high temperatures evolving during acrylate polymerization. Methodical progress in bone marrow trephines embedding in methyl methacrylate with a significant reduction of the polymerization temperature [5, 6] have made immunohistochemistry possible; however, staining results have been weaker or even negative in comparison to paraffin-embedded material after decalcification.…”