Xr60 camera

Whole backskin was isolated from control and mutant newborn pups and fixed in 4% glutaraldehyde, 1 mM CaCl₂, and 0.05 M cacodylic acid (pH 7.4), for 3 h at room temperature and then overnight at 4°C. Samples were washed in 0.1 M sodium cacodylate buffer containing 7.5% sucrose. Samples were postfixed in 1% osmium tetroxide in 0.15 M sodium cacodylate buffer for 1 h and then washed in two changes of 0.11 M veronal acetate buffer for 15 min each. Samples were placed into en bloc stain (0.5% uranyl acetate in veronal acetate buffer) for 1 h, washed in veronal acetate buffer, and then dehydrated in a series of 70, 95, and 100% ethanol. Finally, samples were prepared in 50/50 propylene oxide:Epon resin followed subsequently by two 30-min immersions in 100% Epon resin for embedding. Skin samples were sectioned and imaged with a CM12 transmission electron microscope (Phillips) run at 80 kV with an XR60 camera (Advanced Microscopy Techniques, Woburn, MA). Image acquisition was done using 2Vu software (Advanced Microscopy Techniques). TEM images were visualized using FIJI software. Desmosome morphologies and microseperations were examined in the spinous and granular layers of suprabasal epidermis for all mice. Quantitative analyses of control and mutant epidermis were done for least 15 different regions from two independent mouse litters containing both control and mutant pups.