Laser scanning tcs sp2

EDL muscles from wild‐type (wt) and transgenic (tg) mice were embedded in tissue freezing medium and snap‐frozen in nitrogen‐cooled isopentane. For histological and morphometric analysis, frozen cross sections (7 µm) were stained for hematoxylin and eosin. The distribution and the mean values of CSA, of at least 700 myofibers for each mice group, were analyzed with ImageJ software (Schneider, Rasband, & Eliceiri, 2012). For immunofluorescence analysis, EDL muscle sections were fixed with 4% PFA and processed as described (Dobrowolny et al., 2005). We used the following antibodies: anti‐Laminin, anti‐Myosin Slow, and anti‐Myosin Fast Abs (Sigma‐Aldrich, Saint Louis, MO, USA). Inverted microscope (Axioskop 2 plus; Carl Zeiss Micro Imaging, Inc., Jena Germany) was used, and images were processed using Axiovision 3.1. The number of fast and slow fibers was calculated as percentage of the total number of fibers in the cross section of EDL muscle of the different group of mice. For NMJ analysis, longitudinal frozen sections (40 μm) of quadriceps muscles were stained for fluorescent (BGT‐AF488) α‐bungarotoxin (Thermo Fisher Scientific, Waltham, MA, USA) and confocal images were analyzed using Leica Laser Scanning TCS SP2.

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.

Immunofluorescence experiments were performed on hCPCs cultured on d-HuSk for 4 weeks using primary antibodies specific for actin (α-sarcomeric) (Sigma-Aldrich), Connexin-43 (Abcam, Cambridge, United Kingdom), desmin and dystrophin (Sigma-Aldrich) following a previously published protocol (Castaldo et al., 2013 (link); Nurzynska et al., 2013 (link)). Microscopic analyses were performed with a Nikon Eclipse Ti-E Microscope DS-Qi2 by NIS Elements software (Nikon Instruments, Tokyo, Japan). The expression of cardiac myocyte differentiation markers was also analyzed by confocal microscopy. To this aim, formalin-fixed samples were permeabilized for 3 h with a permeabilizing solution containing 1% BSA and 0.1% Triton in PBS, then blocked with 5% donkey serum and incubated overnight with primary antibodies. Then samples were extensively washed with PBS and incubated for 90 min with a fluorescein-conjugated donkey anti-mouse (Jackson ImmunoResearch Europe). Rhodamin-conjugated phalloidin was used to detect F-actin while TO-PRO3 iodide fluorescent dye 642/661 (Invitrogen) was used for nuclear staining. As a negative control, the primary antibody was omitted. Microscopic analysis and digital microphotography was performed by a Leica Confocal Microscope (Laser Scanning TCS SP2 equipped with Kr/Ar and He/Ne lasers) performing optical spatial series with a step size of 2 μm.