Dmi6000b inverted microscope

Freshly isolated skeletal muscle was frozen in liquid nitrogen-chilled isopentane. 10μm sections were used for all immunostaining. Myofiber cross-sectional area and fiber composition were determined as previously described [29 (link)]. Primary antibodies were purchased from Developmental Studies Hybridoma Bank (type 1 fiber—A4.84, type 2a fiber—SC-71, type 2b fiber—BF-F3, and type 2d fiber– 6H1) and Sigma (laminin—L9393, fast myosin—MY32). A4.84 was also used for the detection of primary myofibers in E18.5 embryos. Secondary antibodies were purchased from Southern Biotechnology (anti-mouse IgM-FITC– 1021–02, anti-mouse IgG-555–1030–32) and ThermoFisher Scientific (anti-rabbit IgG-Alexa Fluor 350 –A11046). Staining of E18.5 immunosections were modified as follows. Slides were thawed on ice for 10 min and then fixed in 2% PFA at room temperature for 10 min. After washing in PBS, the sections were permeabilized with 0.1% Triton X-100 for 5 min, blocked in 15% horse serum in 0.1% Triton/PBS for 1 hour, and then incubated with primary antibodies in blocking buffer overnight at 4C. Slides were mounted with Prolong Gold Antifade Mountant (ThermoFisher Scientific), and visualized with the Leica DMI6000B inverted microscope and the Zeiss Axio Observer Inverted fluorescent microscopes and color CCD camera.

Immunocytochemistry was performed essentially as described and involved cell fixation in 4% paraformaldehyde, permeabilization (except for O4 staining), blocking and consecutive incubation with primary and secondary antibodies, separated by extensive washing cycles (33 (link)). In addition to antibodies already mentioned for their use in immunohistochemistry, the following primary antibodies were used: mouse anti-O4 monoclonal (R&D systems, #MAB1326, Lot# HWW1115081), rabbit anti-Tcf4 (Abcam, #ab185736, Lot#GR3229215-1), rabbit anti-GFP antiserum (Molecular Probes, #A11122, Lot# 1293114), rat anti-MBP monoclonal (Abcam, #ab7349, Lot# GR188102-12 and Bio-Rad, #MCA409S, Lot #210610). Secondary antibodies were coupled to Cy3 (Dianova) or Alexa-Fluor (Molecular Probes) fluorescent dyes. Stainings were documented on a Leica DMI 6000B inverted microscope or a Zeiss Apotome.