Carv 2 confocal imager

As the mutation specifically affects the fast skeletal myosin heavy chain type IIx and as fibre typing for individual cells occurred after the mechanical experiments, we only kept the biophysical data from fibres expressing this specific isoform, i.e., the fast skeletal myosin heavy chain type IIx. Briefly, after the Mant-ATP chase experiments or contractile measurements, individual fibres were stained with an anti-myosin fast/type IIx antibody (IgM isoform, clone 6H1, dilution: 1:50, gift from Dr. Joseph Hoh—University of Sydney, Sydney, Australia) [24 (link)]. Myofibres were then washed in PBS/0.025% Tween-20, and incubated with a secondary antibody conjugated to Alexa 594 in a blocking buffer (from ThermoScientific, Waltham, MA, USA, dilution 1:500). After washing, muscle fibres were mounted in Fluoromount and images taken with a confocal microscope (Zeiss Axiovert 200, objective ×40) equipped with a CARV II confocal imager (BD Biosciences, Franklin Lakes, NJ, USA) [25 (link)]. The Supplementary Figure S1 depicts the pH 4.4 ATPase activity of fibres within the muscles of a control horse, a heterozygote, and an animal homozygous for the MYH1 mutation.

MEFs or MFs seeded on glass coverslips were fixed for 15 min in 3.7% paraformaldehyde, rinsed twice in cold PBS pH 7.4 for 10 min and permeabilized in blocking solution (PBS with 5% BSA and 0.3% Triton™ X-100) for 30 min. Coverslips were then washed twice in chilled PBS pH 7.4 for 10 min and specific primary antibodies (anti-collagen-1 (Calbiochem), α-smooth muscle actin - Cy5 (Sigma-Aldrich), vimentin (Cell Signaling) were diluted 1:250 in antibody dilution buffer: (PBS with 5% BSA and 0.3% Triton™ X-100) were applied overnight at 4 °C. Cells were washed twice in cold PBS pH 7.4 for 10 min, and fluorochrome-conjugated secondary antibody (mouse or rabbit) Alexa Fluor® (Molecular Probes, Life Technologies) diluted 1:2000 in antibody dilution buffer were applied for 2 h at RT in the dark. To visualize DNA, after two 10-min washes, cells were stained with Hoechst 33258 (Molecular Probes, Life Technologies) for 15 min at RT in the dark. The slides were again rinsed in PBS and then the coverslips were mounted on microscope slides using Prolong® Gold Anti-Fade Reagent (Molecular Probes, Life Technologies). Slides were imaged on a BD Biosciences CARV II Confocal Imager.

A Confocal microscope (Zeiss Axiovert 200, objectives ×20 and ×40) equipped with a CARV II confocal imager (BD Biosciences) was used to acquire images. To visualize muscle fibers in 3D, stacks of 200 images were acquired (1 μm Z increments) and analysed with a custom-made MATLAB (MathWorks) program [21 ]. To measure how ordered the nuclear distribution for a particular fiber experimental NN (nearest neighbour) distances were calculated and compared to simulated distributions: a theoretical optimal and a theoretical random distribution. We denoted experimental, optimal and random distributions by M_E, M_o and M_R respectively. An order score g [3 (link)], was then calculated using the following equation: g = (M_E- M_R) / (M_O– M_R).