Bone and cartilage were differentially stained and the body was cleared to enable us to characterize and quantify differences in skeletal morphology associated with offspring size. Bone and cartilage stains were performed following the methods outlined in [7 (link)]. This staining allows for visualization of the internal skeleton and scale development (both total body coverage and scale growth rings). Specimens were visualized using a Nikon dissecting microscope (Nikon SMZ800 dissecting scope and Nikon DXM1200C digital camera).
To determine the degree to which cranial musculature is developing around the time of birth, neonates were immunostained with the muscle-specific antibody MF-20 (DSHB, Iowa, USA), visualized after performing HRP colour reaction (DAB chromogen kit, Biocare Medical, Concord, CA, USA) and the perimeter of the adductor mandibulae traced from a lateral view (for methods see [7 (link)]). Myomere development was also visualized using this colour reaction technique. Specimens were imaged in 1 : 1 glycerol : phosphate buffered saline solution under a dissecting microscope (Nikon SMZ800 dissecting scope and Nikon DXM1200C digital camera).
To determine the degree to which cranial musculature is developing around the time of birth, neonates were immunostained with the muscle-specific antibody MF-20 (DSHB, Iowa, USA), visualized after performing HRP colour reaction (DAB chromogen kit, Biocare Medical, Concord, CA, USA) and the perimeter of the adductor mandibulae traced from a lateral view (for methods see [7 (link)]). Myomere development was also visualized using this colour reaction technique. Specimens were imaged in 1 : 1 glycerol : phosphate buffered saline solution under a dissecting microscope (Nikon SMZ800 dissecting scope and Nikon DXM1200C digital camera).
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