Lsm510 meta confocal microscope

Embryos were collected and stained with various antibodies as previously described (Han and Olson, 2006). The following primary antibodies were used: rabbit anti-Dmef2, 1:1000 (gift from B. Paterson); rabbit anti-Discs large, 1:200 (Developmental Studies Hybridoma Bank); rabbit anti-dystroglycan 1:100 (gift from W.M. Deng). Cy2, Cy3, Cy5 or Biotin-conjugated secondary antibodies (from Jackson Lab) were used. Images were obtained with a Zeiss LSM510-meta confocal microscope or an Olympus BX-51 disc-spin confocal microscope.

To image electrospun NorHA hydrogels, fibers were collected onto aluminum foil and imaged (dry) with a FEI Quanta 600 environmental scanning electron microscope (SEM), or collected onto thiolated coverslips and imaged (hydrated) using confocal laser fluorescent microscopy (Zeiss LSM 510 Meta Confocal Microscope) or wide field fluorescent microscopy (Olympus BX51). To determine fiber diameters, scaffolds were imaged in 6 distinct scaffold areas and fiber diameters were quantified using Image J (>25 fibers per image, 63× magnification – confocal, 9500× magnification – SEM). Fluorescence intensity profiles were generated by drawing a horizontal line across images and analyzing pixel intensity using ImageJ. Fiber angle was quantified by measuring the direction of individual fibers (40× wide field, >20 fibers per image, >150 fibers) in relation to a standard vertical line arbitrarily set at 0 degrees.