ABSTRACT
Half-fibres (split fibres) were prepared from the iliofibularis muscle of young adult Rana pipiens frogs (length ~ 10-11 cm), using an unconventional and powerful technique first described by Endo et al. (1970) and later improved by Horiuti (1986), Villaz et al. (1987) and Vivaudou et al. (1991), with further improvement introduced here. All dissections were performed in a cold room equipped with a dehumidifier (temperature, 4.0°C ± 0.5°C; relative humidity, 25% ± 5%). All muscles were dissected under a stereomicroscope, in Ringer solution containing 116 mM NaCl, 1.65 mM KCl, 1.80 mM CaCl2, 2.15 mM Na2HPO4 and 0.85 mM KH2PO4 (pH ~ 7.2 at 4°C, but very little dependent on temperature) (see Horiuti 1986; Villaz et al. 1987). A single intact fibre was then isolated, under the stereomicroscope, in a relaxing buffer similar to that used by Villaz et al. (1987), consisting of 108.5 mM KMS, 5.4 mM Na2ATP, 10.5 mM Mg(MS)2, 10 mM EGTA and 10 mM PIPES, brought to pH ~ 7.1 at 4°C with KOH, so that the pH was ~7.0 at the working temperature (10°C). Methanesulphonate (MS−) was used for studies of half-fibres (see the next paragraph, pp. 13-14 and many sections concerned with new experimental data presented in this monograph), because of the advantages of this biological material. The dissection process took ~15-20 min. The intact fibre was carefully split lengthwise under the
stereomicroscope (see Vivaudou et al. 1991, particularly their Figure 1 and corresponding comments, for precise details on the technique used to split the fibre). This splitting was carried out in the relaxing buffer, described above, and took ~15 min. Binding to the force transducer (AM 801E, Ackers, Horton, Norway) in the trough and measurements of lengths and apparent diameters of the half-fibres (see pp. 12-13) took ~10 min. Forces were therefore recorded after ~15 min + 10 min ~ 25 min. Force measurements took ~25 min. Thus, the total duration of handling of the half-fibres was ~50 min.
