1.四川大学华西医院/ 华西临床医学院呼吸病学研究室,成都 610041;2.四川大学华西医院临床研究管理部,成都 610041;3.四川大学华西医院呼吸与危重症医学科,成都 610041
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1. Laboratory of Pulmonary Diseases, West China Hospital/ West China School of Medicine, Sichuan University, Chengdu 610041, China. 2. Department of Clinical Research Management, West China Hospital, Sichuan University, Chengdu 610041. 3. Department of Respiratory and Critical Care Medicine, West China Hospital of Sichuan University, Chengdu 610041
目的 优化小鼠肺组织冰冻切片制作方法,提高肺组织冰冻切片质量,有利于提高免疫荧光染色的特异性,获得更准确可靠的实验结果。 方法 使用C57BL/6 小鼠,分别采用传统冷冻后固定法、冷冻前固定法、改良灌注冷冻前固定法3 种方法制作冰冻切片,经免疫荧光染色后使用激光扫描共聚焦荧光显微镜观察肺组织染色情况。使用荧光显微镜对肺组织切片进行全片扫描,计算单位面积内完整气道数量。 结果 传统冷冻后固定法制作的小鼠肺组织冰冻切片,肺泡结构破坏,气道壁断裂严重,存在非特异性染色;冷冻前固定法制作的小鼠肺组织切片,肺泡和气道结构相对完整,但肺泡塌陷,部分气道结构破坏;改良灌注冷冻前固定法制作的小鼠肺组织切片,肺泡和气道结构形态均完整、清晰,多重免疫荧光染色定位准确。改良灌注冷冻前固定法制作的小鼠肺组织冰冻切片单位面积内完整气道(直径≥100 μm)数量高于冷冻前固定法((0. 66±0. 15) / mm2 vs (0. 33±0. 14) / mm2,P< 0. 05),也显著高于传统冷冻后固定法((0. 66±0. 15) / mm2 vs (0. 02±0. 04) / mm2 ,P< 0. 01)。 结论 使用改良灌注冷冻前固定法制作冰冻切片,利于保持小鼠肺组织形态完整,利于获得高质量的多重免疫荧光染色结果。
Objective Optimizing the preparation method to improve the quality of mouse lung cryosections to help enhance the specificity of immunofluorescence staining and obtain more accurate and reliable experimental result. Methods C57BL/6 mouse lung tissue was used to make cryosections via the traditional post-freezing fixation method, prefreezing fixation method, and a modified perfusion pre-freezing fixation method. A laser-scanning confocal microscope was used to observe lung tissue immunofluorescence staining. Whole areas of mouse lung slices were scanned by fluorescence microscope, and the numbers of intact airways per unit area of lung tissue were calculated. Results In the lung cryosections made via the traditional post-freezing fixation method, the alveoli structure was damaged, the airway wall was seriously disrupted, and there was non-specific staining. Lung cryosections made via the pre-freezing fixation method showed relatively intact alveolar and airway structures but collapsed alveoli and several destroyed airways. In the lung cryosections obtained via the modified perfusion pre-freezing fixation method, the structure and morphology of the alveoli and airways were intact and clear. Additionally, the locations of multiple proteins targeted with immunofluorescence staining were accurate. The number of intact airways (diameter ≥100 μm) per unit area in the lung cryosections obtained via the modified perfusion pre-freezing fixation method was higher than that from cryosections made using the pre-freezing fixation method ((0. 66±0. 15) / mm2 vs (0. 33±0. 14) / mm2, P< 0. 05) and was also significantly higher than that from sections made using the traditional post-freezing fixation method ((0. 66 ± 0. 15) / mm2 vs (0. 02 ± 0. 04) / mm2, P< 0. 01). Conclusions The modified perfusion pre-freezing fixation method for cryosections is conducive to maintaining the integrity of mouse lung tissue morphology and obtaining high-quality multiplex immunofluorescence staining result.
叶倩宸,徐 丹,文富强,陈 俊,汪 涛.用于多重免疫荧光染色的小鼠肺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(1):96~102.
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