Eclipse c2 si confocal spectral microscope

Images were acquired on an Olympus DS-Fi2 epifluorescence microscope, using 40X and 100X Olympus UplanFI oil immersion objectives, a Nikon DS-fi2 camera and the Q-Imaging capture software. Confocal acquisition was carried out at Unidad de Microscopía Avanzada (UMA), Pontificia Universidad Católica de Chile. Cells were imaged on a Nikon Eclipse C2 Si Confocal Spectral Microscope with NIS-Elements C software. High-resolution confocal images were acquired at UMA and Massachusetts General Hospital (MGH) Program in Membrane Biology (PMB) Microscopy Core Facility, using the Zeiss LSM 800 Airyscan confocal microscope and Zeiss LSM 880 Airyscan confocal microscope, respectively, with Airyscan acquisition mode and conventional super-resolution processing using the ZEN software by Zeiss.

For skeletal muscle immunofluorescence, cryosections (7μm) were fixed in 4% paraformaldehyde (Merck, Darmstadt, GE), blocked for 1 hour in blocking buffer (1% BSA, BM-0150, Winkler, Santiago, CL), 1% gelatin from cold water fish skin (G7765, Sigma, St. Louis, MO, USA), 0,01% Triton X-100 (X100-1L, Sigma, MO, St. Louis, USA) in PBS, and incubated overnight at 4°C with the following antibodies: anti-fibronectin (Sigma-Aldrich, St. Louis, MO, USA), anti-collagen-I (Abcam, Cambridge, UK), anti-p-Smad3 (Cell Signaling, Danvers, MA, USA), anti-Tcf4 (Cell Signaling, Danvers, MA, USA), anti-PDGFRα (R&D Systems, Minneapolis, MN, USA) anti-laminin (Sigma-Aldrich, St. Louis, MI, USA). The corresponding Alexa Fluor 568 or 488-conjugated anti IgGs (Invitrogen, Carlsbad, CA, USA) were used as secondary antibodies. For nuclear staining, sections were incubated with 1 μg/mL Hoechst 33258 in PBS for 10 minutes [42 (link)]. Slices were then washed in water and mounted in fluorescent mounting medium (DAKO, Santa Clara, CA, USA). Sections were visualized on a Nikon Eclipse E600 epifluorescence microscope or a Nikon Eclipse C2 si confocal spectral microscope using NIS-Elements software v4.20, 32 bit.