Laf af lite software

Intraperitoneal injection of Evans blue dye (EBD) (100 μl of 1% EBD per 10 g of body mass) was performed on a minimum of four animals/strain (24-week-old wild-type, mdx and mdx/IL6 mice), as described (85 (link)). Fluorescent fibers were viewed under an inverted microscope (Axioskop 2 plus; Carl Zeiss Microimaging, Inc.), and images were processed using Axiovision 3.1. and analysed using Scion Image 4.0.3.2. software. Confocal microscopy (Leica Laser Scanning TCS SP2) was used to analyse the total intensity of EBD fluorescence, which represent the full amount of fluorescence held within the entire z-axis of the series, in 4-week-old mdx and 24-week-old mdx whole diaphragm muscles. Approximately 160 optical sections, from at least three separate experiments, were analysed. The images were processed and analysed using LAF AF Lite software (Leica). Acquisition and analysis was performed in a blinded fashion, using coded slides.

The ALI cultures on filter supports were fixed with 3% paraformaldehyde (PFA) and permeabilized with 0.5% Triton X-100. The cultures were blocked with 1% bovine serum albumin (BSA) in phosphate-buffered saline with Tween 20 (PBST) buffer and incubated with primary antibodies, followed by incubation with fluorescence labeled secondary antibodies. The nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole), and ALI cultures were embedded with ProLong Diamond mountant (Life Technologies) on microscope slides. The following antibodies were used: anti-mucin-5AC antibody (Acris), Cy3-labeled antibody against β-tubulin (Sigma-Aldrich), and secondary antibodies conjugated with Alexa Fluor 488 dye (Life Technologies). ALI cultures were analyzed with the Nikon Eclipse Ti microscope and the Leica TCS SP5 confocal microscope. NIS-Elements AR software 3.2 (Nikon), LAF AF lite software (Leica), and ImageJ/Fuji software (National Institutes of Health) were used for image processing. The experiments were repeated at least with six ALI cultures from three independent dogs (n ≥ 6).