Computational protocol: Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy

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Protocol publication

[…] Blood urea nitrogen (BUN) and serum alanine transaminase (ALT) were assayed using kits purchased from Arbor Assays and Cayman Chemicals, respectively. For BUN, serum was diluted 1:20 in ultrapure H2O and analyzed by colorimetry according to the manufacturer’s protocol on a Spectramax microplate reader using Softmax Pro 5.3 software (Molecular devices). ALT was detected in serum diluted 1:10 in ultrapure H2O via consumption of NADH in a coupled reaction. Absorbance measurements were taken at 340 nm (Synergy 2 microplate reader) every minute for 5 min and the average rate of change across a linear range was used to quantitate ALT. [...] Seven-micron tissue cryosections were stained by hematoxylin and eosin (H&E) according to standard protocols. Sections were processed for secondary immunofluorescence (IF) as described previously [, , ]. H&E- or immunofluorescent-stained skeletal muscle sections were imaged using an X71 inverted epifluorescent microscope with a Peltier element-cooled 12.8MP DP72 CCD camera and CellSens software (Olympus).For analysis of eMHC, αDG glycosylation (IIH6) and central nucleation, tiled ×20 images were taken across each entire muscle section and aligned to compile entire section maps in Photoshop (Adobe), then positive and negative fibers were counted manually using ImagePro Express (MediaCybernetics). Data are reported as percentages obtained by dividing the number of positive fibers by the total number of muscle fibers in an entire muscle section. Tissue maps from this analysis were also used for measurement of muscle fiber minimum diameter. Perimeters of individual muscle fibers were traced in a semi-automated fashion using a combination of manual segmentation (dark objects) and manual polygon selection in ImagePro Premier v9.1 (Media Cybernetics), which calculated minimum diameter according to a reference distance (scale bar). Area values were sorted according to size, grouped into bins of 2.5 μm (estimated from the formula h=3.5σn1/3, where h is the optimized bin size, σ is the standard deviation, and n is the number of observations), up to a maximum of 30 μm and plotted as a histogram. Analyses were performed blinded to experimental group and study design.Sections incubated with anti-collagen VI, anti-CD11b, or anti-pS6 were imaged and mapped as above. Whole section maps were analyzed for fluorescent area using Fiji (ImageJ, []) software. Briefly, a region of interest was drawn to encircle the entire muscle section. The number of pixels/section was quantified and a color threshold was set. Pixels at brightness above the color threshold were considered positive for collagen VI, CD11b, or pS6; these pixels were selected and counted. Data are expressed proportion of area, which is the number of positive pixels/total number of pixels in the section for each map. […]

Pipeline specifications

Software tools SoftMax Pro, cellSens, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Muscular Diseases, Muscular Dystrophies, Walker-Warburg Syndrome
Chemicals Tamoxifen, Sirolimus