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Determining the Proportions of Bone and Cartilage Growth in the Crucian Carp (carassius auratus) Using the Modified Simultaneous Differential Staining Technique
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  • Determining the Proportions of Bone and Cartilage Growth in the Crucian Carp (carassius auratus) Using the Modified Simultaneous Differential Staining Technique
  • Determining the Proportions of Bone and Cartilage Growth in the Crucian Carp (carassius auratus) Using the Modified Simultaneous Differential Staining Technique
저자명
Lee. Jin-Heon
간행물명
韓國環境保健學會誌
권/호정보
2010년|36권 4호|pp.337-341 (5 pages)
발행정보
한국환경보건학회
파일정보
정기간행물|ENG|
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이 논문은 한국과학기술정보연구원과 논문 연계를 통해 무료로 제공되는 원문입니다.
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The modified simultaneous differential staining technique, which enables double staining of cartilage and bones, needs to be improved to prevent soft tissues from being damaged during the staining process. Key factors influencing the extent to which soft tissues are damaged include the fixative used, macerating time, potassium hydroxide concentration, incubation temperature and the removal of skin from specimens. Here we describe a protocol that enables the hardening of tissues during bleaching and maceration. We also describe a method for objectively measuring rates of cartilage and bone growth. The use of formalin as a fixative rendered soft tissues more rigid due to the resulting chemical bonds formed between proteins. Blotted specimens were immersed in 1% potassium hydroxide (KOH) and incubated at $37^{circ}C$ for 1 day (smaller specimens) or 2-3 days (larger specimens). The 1% KOH solution was also used as the diluent solution for the subsequent immersion in a graded series of 30%, 50%, 70%, 90%, 100% glycerol solutions, a procedure that made soft tissues even more transparent and hardened. It was not necessary to remove the skin of specimens shorter than 2 cm, since the macerating solution could easily penetrate their thin skin layer and continuously remove those pigments hindering visibility. Since excessive osmosis is another factor that can damage soft tissues in the macerating process by causing the rupture of those cells not able to withstand the osmotic pressure, here it was minimized by balancing the salt concentration between the interior and exterior of cells with the addition of 0.9% sodium chloride (NaCl) in the macerating solution. Finally, to determine the proportions of cartilage and bone growth, photographs of the stained specimens were taken with a dissecting microscope and sections corresponding to the cartilage and bones were cut out from the printed pictures and weighed. Our results show that this method is suitable for the objective evaluation of bone and cartilage growth.