Immunofluorescence signal intensity measurements as a semi-quantitative tool to assess sarcoglycan complex expression in muscle biopsy

Figures

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  Figure 1.. Example of protocol application. A) Representative channel green shows the fluorescent staining for laminin-α2 at 20x magnification; scale bar: 50 μm. B) Conversion on RGB image in 8-bit image. C) Threshold image. D) Representative channel red shows the fluorescent staining for β-SG. E) Conversion on RGB image in 8-bit image. F) Threshold image. G,L) Fiber count with ImageJ plug-in in adult (G) and in a pediatric (L) muscle section stained with laminin-α2. H) Histogram showing the number of counted fibers in adult healthy controls (n=4), in adult LGMDR3 patients (n=3) and in BMD pathological controls (n=7). M) Histogram showing the number of counted fibers in pediatric healthy controls (n=4) and in pediatric LGMDR3 (n=3), LGMDR4 (n=2) and LGMDR5 (n=2) patients. I) Dot plot of laminin-α2 fluorescence signal intensity normalized vs number of counted fibers in adult healthy controls (n=4), adult LGMDR3 patients (n=4) and in BMD pathological controls (n=7). N) Dot plot of laminin-α2 fluorescence signal intensity normalized vs number of counted fibers in pediatric healthy controls (n=4), and in LGMDR3 (n=3), LGMDR4 (n=2) and LGMDR5 (n=2) pediatric patients.

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  Figure 2.. Schematic block diagram summarizing the steps of proposed method.

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  Figure 3.. Comparative analysis of sarcoglycans expression in sarcoglycanopathies, Becker muscular dystrophy (BMD) and controls. Dot plot of fluorescence signal intensity of sarcoglycans normalized vs number of counted fibers in patients, healthy controls and BMD pathological controls. A) a-sarcoglycan, B) b-sarcoglycan, C) g-sarcoglycan and D) d-sarcoglycan.

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  Figure 4.. Dot plot of normalized fluorescence signal intensity of A) a-SG, B) b-SG, C) g-SG, and D) d-SG in the three groups of patients. E) Dot plot of normalized fluorescence signal intensity of laminin-α2 in the same patient’s group. **p≤0.001; *p≤0.05.

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  Figure 5.. A) Dot plot of fibrosis in patients. B) H&E representative images of fibro-fatty tissue deposition in the three groups of patients and control muscle; correlation analysis of residual normalized signal intensity of sarcoglycans and fibrosis; scale bar: 50 μm. C) α-sarcoglycan negative correlation with fibrosis in patients with earlier age of onset. D) d-sarcoglycan negative correlation with fibrosis in patients with 10-18 years as age of onset. E) d-sarcoglycan negative correlations with fibrosis in patients with later age of onset.

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  Figure 6.. A) Representative immunofluorescence images of laminin-α2 and sarcoglycans in LGMDR3 patient; scale bar: 50 μm. B) Representative Western blot for sarcoglycans in one LGMDR3 patient. Myosin was use as internal standard. C) Regression line of single immunohistochemistry and Western blot date. D) Bland-Altman plot.