骨骼肌组织冰冻切片制备方法优化研究
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山西医科大学法医学院,山西 晋中 030600

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R-33


Optimal method for preparing frozen skeletal muscle tissue sections
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Affiliation:

Department of Forensic Medicine, Shanxi Medical University, Jinzhong 030600, China

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    摘要:

    目的 探索新鲜和固定骨骼肌组织的最优冰冻切片方法,为骨骼肌疾病的快速诊断及骨骼肌疾病发病机制的研究奠定实验基础。 方法 提取C57BL/6J小鼠胫骨前肌,新鲜组织经液氮直接速冻、包埋剂联合液氮速冻、异戊烷联合液氮速冻前处理;固定组织经直接包埋、包埋剂联合液氮速冻进行前处理,制作冰冻切片后HE染色,并计算冰晶和肌纤维横截面积以评估不同前处理方式的切片效果。 结果 新鲜组织经液氮直接速冻和异戊烷联合液氮速冻处理后均可见肌纤维束形态消失,大量的冰晶空泡;包埋剂联合液氮漂浮速冻,可见肌纤维束完整致密、无冰晶,为适合新鲜组织的前处理方法。固定组织包埋剂联合液氮速冻处理后肌纤维束完整、无冰晶。 结论 新鲜和固定组织经包埋剂联合液氮速冻处理后肌纤维束完整、无冰晶,便于免疫组化、免疫荧光等实验的进一步进行,有利于准确快速地诊断骨骼肌疾病及探索骨骼肌疾病的发病机制。

    Abstract:

    Objective To explore the optimal method for preparing fresh and fixed skeletal muscle tissues, and to lay an experimental foundation for the rapid diagnosis of and research into the pathogenesis of skeletal muscle diseases. Methods The tibialis anterior muscle was extracted from C57BL/6J mice. Fresh tissue was treated by direct rapid freezing in liquid nitrogen, embedding combined with liquid nitrogen freezing, and foreign body alkane treatment combined with liquid nitrogen freezing. Fixed tissues were pre-treated by direct embedding with embedding agent combined with rapid liquid nitrogen freezing. The frozen sections were stained with hematoxylin and eosin. The cross-sectional areas of ice crystals and muscle fibers were calculated to evaluate the effects of the different pre-treatment method. Results The morphology of the muscle fiber bundles was disrupted and numerous ice crystal vacuoles were observed in fresh tissues after direct liquid nitrogen freezing and foreign body alkane treatment combined with liquid nitrogen freezing. In contrast, the muscle fiber bundles were intact and dense and there were no ice crystals in tissues treated with embedding agent combined with rapid liquid nitrogen freezing, indicating that this pre-treatment method was suitable for preparing fresh skeletal muscle tissue. Fixed tissue treated with embedding agent and liquid nitrogen freezing also showed complete muscle fiber bundles and no ice crystals. Conclusions Treatment of fresh and fixed skeletal muscle tissues with embedding agent combined with rapid liquid nitrogen freezing preserves muscle fiber bundles, with no ice crystals. Tissues prepared by this method are thus suitable for further examinations, such as immunohistochemistry and immunofluorescence. This method will therefore aid the accurate and rapid diagnosis of and research into the pathogenesis of skeletal muscle diseases.

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张洁云,芦星宇,刘晋芳,曹锡梅,李 娜,孙俊红,梁新华.骨骼肌组织冰冻切片制备方法优化研究[J].中国比较医学杂志,2024,34(10):64~71.

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  • 收稿日期:2024-04-10
  • 在线发布日期: 2024-11-19
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