Antibodies used on muscle sections.

<p>Antibodies used on muscle sections.</p

Dystrophin quantification in a population of myofibres identified in entire muscle sections performing the double labelling anti-dystrophin ab15277 and anti-spectrin.

<p>Representative images of entire muscle sections stained and acquired by the Axio Scan slide scanner and processed with Definens algorithm derived from a control (a) and from a DMD patient (b). Graph of representative dystrophin quantification in the fibre population of a control, a DMD and a BMD muscle sample (c). Dystrophin quantification was plotted as cumulative fibre count (%) on the primary y axis. The blue dashed line represents the dystrophin intensity distribution of a representative control sample (control 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t001" target="_blank">Table 1</a>), the green dashed line represents the BMD sample and the DMD sample is represented by the red dashed line. The distribution curve of the absolute dystrophin fibre count for the control sample is represented by the blue peak, for the BMD is expressed by the green peak and the DMD fibre population curve is represented by the red peak. Fibres analysed in this representative muscle sections were: ~ 14500 for the control, ~ 10000 for the BMD and ~ 2700 for the DMD muscle section.</p> <p>DMD: Duchenne Muscular Dystrophy; BMD: Becker Muscular Dystrophy; i.v: intensity values.</p

Dystrophin expression in different muscle types obtained from paediatric controls.

<p>Ten muscle blocks derived from different muscle groups obtained from seven controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t001" target="_blank">Table 1</a>) were analysed performing anti-dystrophin ab15277/anti-spectrin and anti-dystrophin MANDYS106/anti-laminin stainings. Entire muscle sections were acquired by the Axio Scan slide scanner and processed by the Definiens algorithm, exploiting spectrin or laminin staining for fibre identification. Dystrophin was quantified in each individual fibre using either anti-dystrophin ab15277 (Fig 4a) or MANDYS106 (Fig 4b) intensities plotted as arbitrary units. We acquired a different number of myofibres (22,000 fibres for control 3 to 5,000 fibres for control 5) per section depending on the cross sectional area.</p

Intensity dystrophin range in muscle sections of DMD/BMD patients and controls.

<p>Intensity dystrophin range in muscle sections of DMD/BMD patients and controls.</p

Labelling variability in different biological replicates of controls.

<p>Labelling variability in different biological replicates of controls.</p

Algorithm development for identifying myofibres.

<p>Spectrin or laminin allow fibre identification within the muscle section. The algorithm was instructed to recognize the tissue within the image (a) and to subsequently recognize the myofibres within the section (b). The algorithm then generated a mask considering only structures presenting fibre characteristics (i.e. excluding nerves, spindles, blood cells, folded muscle tissue and connective areas (c). The mask generated by the algorithm included only the sarcolemma compartment of myofibres and clearly defined fibre rims (d).</p

Dystrophin quantification in a population of myofibres identified in an entire muscle sections performing the double labelling anti-dystrophin MANDYS106 and anti-laminin.

<p>Representative images of entire muscle sections stained and acquired by the Axio Scan slide scanner and processed with Definiens algorithm derived from a control (a) and from a DMD patient (b). Representative dystrophin quantification in population of fibres of control, DMD and BMD muscle samples (c). Blue dashed line represents the dystrophin intensity distribution of a representative control sample (control 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t001" target="_blank">Table 1</a>). Dystrophin absolute count obtained from the BMD sample (BMD 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t001" target="_blank">Table 1</a>) is represented by the dark green dashed line whereas the related dystrophin intensity distribution of the DMD sample is represented by the pink dashed line. The distribution curve of the absolute dystrophin fibre count for the control sample is represented by the blue peak, for the BMD sample is expressed in dark green and the DMD sample is represented by the pink peak. Numbers of fibres analysed in these representative muscle sections were: ~ 1300 for the control, ~ 10000 for the BMD and ~ 2700 for the DMD muscle section.</p> <p>DMD: Duchenne Muscular Dystrophy; BMD: Becker Muscular Dystrophy; i.v: intensity values.</p

Spectrin and laminin mean intensity in muscle sections of DMD, BMD and control patients.

<p>Muscle sections were cut from two paediatric controls, two DMD and two BMD patients. Two biological replicate experiments were performed and for each experiment two sections per sample were stained with anti-spectrin or anti-laminin and acquired by the Axio Scan slide scanner. The Definens script extrapolated spectrin or laminin intensities for each individual myofibre identified within the section. Protein intensities data for spectrin (a) or for laminin (b) were grouped per category (CTR, DMD and BMD) as mean±SEM. The Mann Whitney test revealed significant differences between CTR, DMD and BMD for both spectrin (a) and laminin (b) fluorescent intensities (***, p<0.001). (CTR: control; DMD: Duchenne muscular dystrophy; BMD: Becker muscular dystrophy; SEM: standard error of the mean).</p

Variability in dystrophin quantification in independent immunostaining and dystrophin signal detection stability across the time.

<p>Double staining with different antibody combinations were performed (anti-dystrophin ab15277/anti spectrin (a) and anti-dystrophin MANDYS106/anti-laminin (b)). Sections were cut and stained immediately (A). Muscle blocks were kept at -80°C for one month and then two section sets were cut and again stained immediately. One set of these stained sections were acquired immediately (B) whereas the other stained section set was kept at 4°C for three months and then acquired (C) in order to evaluate the fluorescent signal stability of dystrophin staining after slides long time storage. Dystrophin intensities were quantified per each individual fibre and were plotted as scatter plots (mean±SEM) in arbitrary units (***p< 0.001, Mann Whitney test). Dystrophin intensity dynamic range and number of fibres acquired per experiment are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t003" target="_blank">Table 3</a>.</p

Dystrophin and sarcolemma proteins intensities used in single and double labelling.

<p>Serial sections obtained from two paediatric control muscles (control 1 and 2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194540#pone.0194540.t001" target="_blank">Table 1</a>) were used in single or double labelling. (Single labelling: anti dystrophin ab15277, or anti dystrophin MANDYS106; double labelling: anti dystrophin ab15277 combined with anti spectrin, or anti dystrophin MANDYS106 combined with anti laminin). For each staining, two sections were stained simultaneously and four images were acquired per section using a fluorescent microscope and analysed by Metamorph software following the Arechavala et al., 2010 method. Sarcolemma intensity values were plotted on the y axis (Mean±SEM). Dystrophin intensity values plotted as dots are the difference between dystrophin intensity at the sarcolemma and dystrophin intensity within the cytoplasm (considered to be background staining signal). Analyses were performed using anti-dystrophin ab15277 (a), anti-dystrophin MANDYS106 (b), anti-spectrin (c) and anti-laminin (d).</p