Materials and Methods
Mice obtained for this study were products of pudgy breeding pairs from Jackson Laboratories, Bar Harbor, Maine. For the affected pudgy mice, heterozygous unaffected littermates served as controls. An affected pudgy mouse (pu/pu) can be identified at birt
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Materials and Methods
2.1
Source, Distribution, and Ages of Pudgy and Non-affected Mice
Mice obtained for this study were products of pudgy breeding pairs from Jackson Laboratories, Bar Harbor, Maine. For the affected pudgy mice, heterozygous unaffected littermates served as controls. An affected pudgy mouse (pu/pu) can be identified at birth since it is approximately three-quarters the length of its non-affected littermates (pu/+) and has a markedly shortened, twisted tail. The mice were sacrificed by intraperitoneal injections of sodium pentobarbital. Vertebral and rib assessments were performed in 68 mice, 37 affected (pu/pu) and 31 non-affected (pu/+) age-matched siblings from the late embryo to 3 months of age. There were eight sets of births (litters) in which two or more of the sibling littermates were affected, allowing for a comparison of rib and vertebral anomalies in pudgy mice from the same mother and same pregnancy as well as with all other pudgy mice.
2.2
Whole Mount Preparations
Twelve mice underwent whole mount preparation, seven pu/pu and five pu/+. Following sacrifice the skin, heart, lungs, and abdominal contents were removed. Extremity muscles were not excised. The specimen was exposed to running cool tap water for 1 h, dried gently with paper towels, and placed in a fixative-stain solution composed of absolute alcohol (80 ml), acetic acid (20 ml), and alcian blue 8GX (Sigma Chemical Co., St. Louis, MO) (15 mg). The specimen was left in this solution for 24–48 h, the length of time being dependent on the intensity of alcian blue staining of cartilage portions of the skeleton. Following this the specimen was dehydrated in absolute alcohol for 5 days. Further staining was performed with © Springer International Publishing Switzerland 2016 F. Shapiro, Disordered Vertebral and Rib Morphology in Pudgy Mice, Advances in Anatomy, Embryology and Cell Biology 221, DOI 10.1007/978-3-319-43151-2_2
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2 Materials and Methods
alizarin red (Sigma) prepared using 10 mg alizarin red in 1 % potassium hydroxide. The staining continued until a deep red color indicated those skeletal parts that had converted to bone tissue. The specimen was then transferred for clearing to Mall solution composed of 79 ml water, 20 ml glycerin, and 1 g of potassium hydroxide. Once clearing of the adjacent soft tissues had been completed, the specimen was transferred to glycerin in increasing amounts from 70 to 80 to 90 %. The specimen was stored eventually in 100 % pure glycerol at room temperature in a closed drawer to minimize exposure to light that could lead to fading of the stain. Examination was performed to assess vertebral and rib patterns from dorsal, volar, and right and left lateral planes using a dissecting microscope (Carl Zeiss, Germany) equipped with a Contax RT5 electronic SLR system camera (Yashica, Japan). This method was adapted from those described previously [8–10].
2.3
Radiographic Studies
The vertebral column and ribs (primarily adjacent posterior and lateral ribs) were assessed using specimen r
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