Orca flash 2.8 digital cmos camera c11440

Primary fibroblasts were pulse-labelled with BrdU (10 µM final concentration) for 1 h at 47 h after initial treatment. For assessment of cell proliferation, cells were fixed with 4% paraformaldehyde (PFA) (15713-S) (Electron Microscopy Sciences, PA, USA) for 10 min. DNA was denatured using 2 N HCl containing 0.5% Triton X-100 (9410) (Merck Millipore, MA, USA) for 30 min at 37 °C followed by neutralization with 0.1 M borate buffer pH 8.5 for 10 min. Nonspecific staining was blocked with 5% normal goat serum (PCN5000) (Invitrogen, CA, USA) for 30 min before applying mouse monoclonal anti-BrdU antibody (1:1000, B2531) (Sigma-Aldrich, MO, USA) for 1 h. Thereafter, goat anti-mouse Alexa Fluor® 568 IgG (H + L) (1:500, A-11004) secondary antibody (Invitrogen, CA, USA) was incubated for 1 h, postfixed the cells with 4% PFA for 5 min, and stained with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) (D1306) (Invitrogen, CA, USA) for nuclear visualization. Representative images were acquired using an Olympus Inverted Fluorescence Microscope Model IX83 (Olympus, Tokyo, Japan) equipped with ORCA-Flash 2.8 Digital CMOS Camera (C11440) (Hamamatsu Photonics, Hamamatsu, Japan). Ten images were randomized at magnification × 100 for quantitative analysis of % BrdU⁺ nuclei/total nuclei using image acquisition software (cellSens Dimension Desktop, Olympus, Japan).

The myotubes were fixed with 4% paraformaldehyde (PFA), which was prepared from 20% PFA aqueous solution (15713‐S; Electron Microscopy Sciences, PA, USA) for 10 min, permeabilized with 0.1% Triton X‐100 (9410; Merck Millipore) for 5 min, and blocked with 5% normal goat serum (PCN5000; Invitrogen, Rockford, IL, USA) for 30 min. Primary antibody to detect myosin heavy chain (MHC) (05‐716; Upstate, CA, USA) at a 1:1000 dilution was incubated at room temperature (RT) for 1 h and at 4°C overnight. Thereafter, the cells were incubated with a 1:500 dilution of goat anti‐mouse Alexa Fluor 568 (A11004; Invitrogen) for 2 h at RT in the dark and counterstained with 4′,6‐diamidino‐2‐phenylindole, dihydrochloride (D1306; Invitrogen) for 5 min to visualize the myonuclei. Images were acquired using an Olympus Inverted Fluorescence Microscope Model IX83 (Olympus, Tokyo, Japan) equipped with an ORCA‐Flash 2.8 Digital CMOS Camera (C11440) (Hamamatsu Photonics, Hamamatsu, Japan), and at least 15 images per well were randomly captured at ×100 magnification. A minimum of 10 nuclei containing myotubes positively stained with MHC were selected, and at least 200 myotube diameters per well were included in the analysis using ImageJ software; these myotubes were obtained from 15 randomly separated fields in three independent experiments (National Institutes of Health [NIH], Bethesda, MD, USA).

Cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100, and blocked for non-specific staining with 5% normal goat serum. The primary and secondary antibodies were used as follows: mouse monoclonal anti-MyoD (G-1) antibody (1:500, sc-377460), mouse monoclonal anti-myogenin (F5D) antibody (1:200, sc-12732), mouse monoclonal anti-VDR (D-6) (1:200, sc-13133), rabbit polyclonal anti-CYP24A1 (1:500, sc-66851), and rabbit polyclonal anti-CYP27B1 (1:500, sc-67261) (Santa Cruz Biotechnology, CA, USA); mouse monoclonal anti-myosin heavy chain antibody (MHC) (1:500, 05-716) (Upstate, CA, USA); goat anti-mouse Alexa Fluor^® 488 IgG1 (1:500, A-21121), goat anti-mouse Alexa Fluor^® 568 IgG2a (1:500, A-21134), goat anti-mouse Alexa Fluor^® 488 IgG2b (1:500, A-21141), goat anti-mouse Alexa Fluor^® 568 IgG (H + L) (1:500, A-11004), and goat anti-rabbit Alexa Fluor^® 488 IgG (H + L) (1:500, A-11008) (Invitrogen, CA, USA). Nuclei were stained with DAPI to delineate nuclear localisation. Representative images were taken at ×200 and ×400 magnifications using an Olympus Inverted Fluorescence Microscope Model IX83 (Olympus, Tokyo, Japan) equipped with ORCA-Flash 2.8 Digital CMOS Camera (C11440) (Hamamatsu Photonics, Hamamatsu, Japan).