Control of brittleness in butyl-methylmethacrylate resin embedding mixtures to facilitate their use in immunofluorescence microscopy.
Butyl-methylmethacrylate resin mixtures were tested for brittleness-inducing factors in polymerised resin using a rapid quantitative scoring technique. The major source of brittleness was identified as the reducing agent dithiothreitol, which is commonly included in resin mixtures at 10 mM, to protect against tissue oxidation. Lowering the dithiothreitol content to 5 mM substantially reduced brittleness. Changing the 4:1 ratio of butyl- to methylmethacrylate to 9:1 or 3:2, and reducing the concentration of the catalyst, benzoin ethyl ether, to 0.25% also reduced dithiothreitol-induced brittleness. Polymerisation at temperatures close to 0 degrees C increased dithiothreitol-induced brittleness, but this was controlled in the 4:1 and 9:1 resin mixtures by lowering the catalyst concentration from 0.5 to 0.25%. Degassing the resin mixture with nitrogen gas prior to polymerisation did not reduce brittleness. Immunolabelled onion roots which were embedded using the 3:2 resin mixture ratio, 5 mM dithiothreitol and the 0.25% catalyst concentration, showed excellent preservation of cortical microtubule arrays.
Palmer
J H
JH
School of Biological Science, The University of New South Wales, Sydney, Australia.
Harper
J D
JD
Marc
J
J
D004229:Dithiothreitol / Q000494:pharmacology; D005455:Fluorescent Antibody Technique / Q000379:methods; D008768:Methylmethacrylates / Q000138:chemical synthesis* / Q000737:chemistry / Q000592:standards; D008856:Microscopy, Fluorescence / Q000379:methods; D019697:Onions; D018517:Plant Roots / Q000648:ultrastructure; D018583:Pliability / Q000187:drug effects; D012116:Resins, Plant / Q000138:chemical synthesis* / Q000737:chemistry / Q000592:standards*; D016610:Tissue Embedding
D008768:Methylmethacrylates; D012116:Resins, Plant; C060877:Dakril 4B; D004229:Dithiothreitol