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Mercuric chloride in alcohol and chloroform used as a rapidly acting fixative for contracting muscle fibres
Author(s) -
Brown L. M.,
Hill L.
Publication year - 1982
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1982.tb00348.x
Subject(s) - fixative , glutaraldehyde , myofibril , fixation (population genetics) , uranyl acetate , chemistry , biophysics , protein filament , sarcomere , anatomy , chromatography , ultrastructure , biochemistry , biology , myocyte , cytoplasm , gene , endocrinology
SUMMARY A fixative which preserves the correct alignment of the filaments in actively contracting muscle fibres must prevent sliding during fixation. Glutaraldehyde is unsuitable because the fibres are capable of relaxation and contraction for several seconds. A modification of Carnoy's fixative rapidly fixes tetanized fibres without relaxation or further activity. The fixative contained ethanol and chloroform saturated with mercuric chloride. Fibres were thereby fixed and dehydrated at the same time. They were then stained with uranyl acetate, embedded, and stained with lead in the usual way. Isolated fibres from R. temporaria were tetanized with low voltage pulses in air, and normal tetanic tensions were recorded prior to fixation. At least 50% of the tension remained after fixation. Unstimulated fibres were activated by the fixative, but fibres depolarized with K+ developed no tension. Embedded fibres were examined intact in polarized light and closely resembled fresh fibres. Electron micrographs showed that, although the membranes were disrupted, the myofibrils were well preserved to 15 μm below the surface and the band pattern was maintained throughout the fibre. There was some local variability in the width of the overlap zone, especially where myofibrils branched. The fine structure of the filaments was similar to that seen after conventional fixation. The cross‐bridges were visible, but the alcoholic dehydration probably interfered with their orientation. Measurements of filament length, and of troponin and cross‐bridge periods by optical diffraction of calibrated electron micrographs, indicated differential shrinkage between the I‐band, overlap zone and H‐zone similar to that seen with other fixatives. Criteria for satisfactory fixation of contracting muscle are discussed. The fixative is considered to be suitable for examining the longitudinal alignment of the filaments, and also for periodicity measurements if the small shrinkage artefact is allowed for. It is unsuitable for studying the reticulum, or for measuring the volume ratio of the myofibrils.