Leica dfc360 fx

For karyotyping, the chromosome slides were stained using fluorescent DNA dyes, either 4',6-diamidino-2-phenylindole (DAPI) and / or chromomycin A3 (CMA), according to standard protocols [22 ]. Note that CMA and DAPI are GC- and AT-specific, and thus lead to stronger staining of GC- and AT-rich regions, respectively [23 ]. For microdissection (see below), the chromosome slides were stained with 0.1% Giemsa stain (Sigma) for 3–5 min at RT.
Images of fluorescently stained metaphase chromosomes were captured using either (i) a CCD-camera installed on a Axioplan 2 compound microscope (Carl Zeiss, Germany) equipped with filtercubes #49, #10, and #15 (ZEISS, Germany) using AxioVision (Carl Zeiss, Germany) or ISIS4 (METASystems GmbH, Germany) at the Multiple-access Center for Microscopy of Biological Subjects (Institute of Cytology and Genetics, Novosibirsk, Russia); or (ii) a Leica DFC 360 FX camera (Leica Microsystems CMS GmbH) installed on a Leica DM 2500 compound microscope with filtercube B/G/R (Leica Microsystems) using LAS V4.1 (Leica Microsystems) at the Zoological Institute, University of Basel, Switzerland.

To block nonspecific binding sites, cells were incubated with a 10% solution of normal goat serum (Sigma, St. Louis, MI, USA) in PBS with the 1% bovine serum albumin BSA (PanEco) for 1 h at RT. Then, the samples were incubated with a solution of anti-CD206 antibodies (ab64693, Abcam, 1:100) or rabbit polyclonal control IgG (910801, Biolegend) as a control for 2 h at RT, and subsequently with Goat anti-Rabbit antibody conjugated with Alexa594 (A11037, Invitrogen, 1:1000). The nuclei were labeled with DAPI (Sigma, St. Louis, MI, USA). Samples were analyzed with a Leica DM6000B fluorescent microscope equipped with a Leica DFC 360FX camera (Leica Microsystems GmbH, Wetzlar, Germany).

Images were viewed at 20× magnification (Leica DM2500, Leica Microsystems, Milton Keynes, UK) and captured with a digital camera (Leica DFC360 FX, Leica Microsystems). At least 50 type I and 75 type II muscle fibers were counted to ensure accurate assessment of the muscle fiber-specific satellite cell content (Mackey et al. 2009 (link)). In this study, 63 ± 4 type I and 92 ± 6 type II muscle fibers were evaluated for each muscle biopsy per participant. Image processing and quantitative analyses were completed using ImageJ version 1.49 software (Schneider et al. 2012 (link)). All quantitative analyses were conducted in a blinded fashion to the participant coding and experimental trial. The identification of Pax7⁺ and MyoD⁺ cells were determined by the co-localisation of either Pax7 or MyoD with DAPI, and located at the periphery of each muscle fiber. Double stained muscle cross sections (first stained for satellite cell and then fiber type) were superimposed to determine the satellite cell response in a muscle fiber-specific manner. This is important as previous studies have shown changes in RE-induced satellite cell response occur in a muscle fiber type-specific manner (Snijders et al. 2012 (link); Cermak et al. 2013 (link)).