Table 1.
Control, BMD, and DMD muscle samples used.
Table 2.
Antibody reagents for immunofluorescence detection of dystrophin and spectrin.
Figure 1.
Immunofluorescence analysis of dystrophin intensities per fiber in a muscle fiber population.
A. Cryosections of a healthy control muscle sample (control 3, quadriceps muscle) co-stained with anti-dystrophin antibody MANDYS106 and an anti-spectrin antibody. The sections were imaged using a confocal microscope, with the green channel (Alexa488) showing dystrophin and the red channel (Alexa594) showing spectrin. B. Images were analyzed using customized Definiens software, which automatically identifies individual muscle fiber membranes using their spectrin signal and measures the membrane dystrophin intensity per muscle fiber. C. Dystrophin intensity distribution in muscle fiber population analyzed, shown as a regular histogram (blue line) or a cumulative histogram (red line). (au: arbitrary units).
Figure 2.
Dystrophin expression in healthy (non-DMD) control muscle samples from different muscle groups and different donors using MANDYS106.
Eight muscle samples were compared within one experiment: six muscle groups from one donor (control 1) and two muscle groups from two other donors (controls 2 and 3). Analysis was performed on two sections per sample; imaging was performed with microscope settings suitable for imaging control samples (1% laser intensity). A. Representative immunofluorescence images of different muscle groups from Controls 1–3. B. Summary of numbers of fibers analyzed per samples, morphology parameters (fiber cross-section surface area and membrane thickness), mean spectrin intensities and dystrophin intensity parameters (mean, Q90 mean and maximum) of all the samples analyzed. C. Dystrophin intensity distribution in the fiber populations (cumulative plots) of three different muscle groups analyzed for control 1. (au: arbitrary units; CV%: coefficient of variation of replicate sections; CTRL: control; DMD: Duchenne muscular dystrophy; SD: standard deviation).
Figure 3.
Dystrophin intensity in control muscle samples, and pre-treatment DMD and BMD muscle biopsies.
A. Representative immunofluorescence images (1 of 10 images) from the tibialis anterior muscle samples (from one control subject (CTRL2), two patients with DMD and four patients with BMD. Analysis was performed in the same experiment using the MANDYS106 antibody and ‘control’ imaging settings (1% laser intensity). B. Corresponding dystrophin membrane intensity distribution in the fiber populations analyzed (cumulative graphs). (au: arbitrary units; BMD: Becker muscular dystrophy; CTRL: control; DMD: Duchenne muscular dystrophy).
Table 3.
Reproducibility of detecting differences in dystrophin expression in muscle biopsies with a wide range of dystrophin levels from healthy controls to BMD and DMD samples by immunofluorescence using MANDYS106 antibody and Definiens intensity measurements (at ‘control’ microscope settings).
Figure 4.
Dystrophin signal specificity using MANDYS106.
Immunofluorescence intensities measured in pre-treatment biopsies of different patients with DMD at microscope settings suitable for imaging DMD samples (7% laser intensity). A. (Left): Dystrophin staining (AlexaFluor488). All fibers exhibit varying intensities of dystrophin (trace dystrophin) and certain fibers termed “revertants” express higher levels of dystrophin. (Middle): Spectrin staining (AlexaFluor594): Fibers with equivalent spectrin intensities exhibited varying levels of dystrophin staining. (Right): Histogram of the mean dystrophin intensity in the fiber population depicting the fibers expressing trace dystrophin and revertant fibers. The mean dystrophin intensity of the fiber population expressing trace dystrophin is indicated by the arrow. B. Specificity of MANDYS106 antibody compared with isotype control for measuring dystrophin expression. Immunofluorescence images of MANDYS106 (left) or negative control isotype staining (right) of patients DMD 4 (deletion exon 45) and DMD 6 (deletion exon 43), and the corresponding histograms of mean dystrophin intensity (cumulative graphs). C. Dystrophin intensities in patients with DMD with different exon deletions (DMD patient 1: deletion exon 48–50; DMD patient 3: deletion exon 50; DMD patient 5: deletion exon 45; DMD patient 6: deletion exon 45). These samples were analyzed in the same experiment. (au: arbitrary units; del: deletion; DMD: Duchenne muscular dystrophy).
Table 4.
Reproducibility of detecting differences in dystrophin expression in DMD muscle biopsies by immunofluorescence using MANDYS106 antibody and Definiens intensity measurements (at high laser settings).
Table 5.
Summary of experiments: different operators performing the staining, imaging, and Definiens image processing.
Figure 5.
Dystrophin intensities in pre- and post-treatment biopsies from DMD patient 4 (deletion exon 45) using two different anti-dystrophin antibodies.
A. Representative immunofluorescence images (1 of 10) and dystrophin intensity measurements of the muscle fiber population of pre- and post-treatment biopsies using MANDYS106 (left) and the corresponding cumulative graphs (right). B. Representative immunofluorescence images (1 of 10) and dystrophin intensity measurements of the muscle fiber population of pre- and post-treatment biopsies using ab15277 (left) and the corresponding cumulative graphs (right). C. Reproducibility of the detection of dystrophin intensity difference between post-treatment and pre-treatment biopsies using MANDYS106 and ab15277. Percentage dystrophin difference = ([dystrophin intensity post-treatment – dystrophin intensity pre-treatment]/dystrophin intensity pre-treatment ×100). The statistical significance was calculated using a mixed model analysis taking into account the following covariance parameters: variance between images of one section, variance between sections, variance between slides. The mean difference was significant at the 0.05 level. No difference was observed for the spectrin staining. (au: arbitrary units; CV: coefficient of variation; N/D: not determined).