Enzyme histochemistry on bone marrow sections after embedding in methacrylate at low temperature

Summary This paper presents a method for the application of light microscopy to enzyme histochemistry on semi-thin sections of non-decalcified bone marrow cylinders (4×15 mm), entire rat femurs and larger soft-tissue specimens (4×30 mm²) after embedding in a methacrylate mixture which is then polymerized at 4°C. The best results were obtained using 1–4 h fixation in 4% formaldehyde solution in 0.1 M cacodylate buffer and propanol, . Dehydration of the tissue in concentrated solutions of propanol was complete within 2 h. It was then impregnated for 4 h in the embedding medium containing 55% 2-hydroxyethyl methacrylate, 27% methyl methacrylate, 9% 2-hydroxyethyl acrylate, 9% propanol and 2% di-benzoyl peroxide. For the final polymerization the amount of peroxide was reduced to 0.4%, and 0.1% N,N-dimethylaniline was added as a co-initiator. Polymerization lasted about 10 h at 4°C; the heat of the reaction caused the internal temperature to rise, reaching a peak of 20°C after 6 h. The blocks could then be inserted directly into the tissue-holder of a rotation microtome and cut with a steel knife. Semi-thin sections (1–3 m), free from wrinkles, were easily obtained: on the surface of 1% acetic acid at 35°C, even bone-containing sections spread out spontaneously. Enzyme activity was well preserved throughout the entire section when tested for acid (acPase) and alkaline phosphatase (alkPase), non-specific esterase (nEst), butyrate esterase (bEst), -naphthol-AS-D-chloroacetate esterase (chEst), and peroxidase (poAse) on entire rat femurs and bone marrow biopsy cylinders of different species including human. Enzyme activity was still present in the blocks after a 2,5-years storage time. AlkPase outlined a network of reticulum cells, and marked osteoblasts and granulocytic cells (human). AcPase activity was strong in osteoclasts, macrophages, and Golgi zones of megakaryocytes and myeloid precursors. Best clearly marked monocytes and fat cells (rat), but not bone marrow macrophages, nEst followed the pattern of AcPase. PoAse was seen in the osteolytic rim of osteoclasts and in granulocytes. Treatment of the sections with 20% sucrose prior to incubation broke the latency of acPase and alkPase after overfixation.