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冷冻切片的优点是能完好保存有机溶剂及热敏感抗原,其缺点是制片过程中形成冰晶影响免疫组织化学检测结果。为了解决这一难题,我们对现有的冷冻切片法进行了改良。多聚甲醛固定和蔗糖脱水后的脑、肺和睾丸组织,在包埋和切片之前,利用OCT包埋剂(聚乙二醇和聚乙烯醇水溶性混合物)进行预包埋,切片后进行苏木精伊红染色(hematoxylin eosin staining, HE染色)和免疫组织化学染色(Immunohistochemical staining, IHC染色),并比较了改良冷冻切片法、常规冷冻切片法和石蜡切片法制作的切片HE和IHC染色效果。结果显示,切片前使用OCT包埋剂充分浸润组织6～48 h,可显著地提高冷冻切片质量;与常规冷冻切片法相比,用改良法制作的睾丸和脑组织冷冻切片结构更加清晰,因冰晶形成的空洞较少,总体质量均得到极大提高;与传统石蜡切片相比,改良法制作的肺组织冷冻切片质量尤佳。本研究建立了一种适用于免疫组织化学研究的冷冻切片新方法,无需使用液氮骤冷,大幅降低了多组织样品制片的难度,并且获得了正确的IHC染色结果,从而简化了实验流程,为组织形态学研究和抗原检测提供了可靠手段。
Abstract：Cryosections can preserve organic solvent and thermosensitive antigens,but the formation of ice crystals during sectioning often exerts negative impact on immunohistochemical(IHC) staining.In order to fight the problem,the conventional cryosectioning was modified here.The tissue blocks from chicken brain,lungs,testis were immobilized with paraformaldehyde,dehydrated with sucrose,and pre-embeded with optimal cutting temperature compound(OCT) for prior to OCT embedding and sectioning.The paraffin sections,modified cryosections and conventional ones were under gone hematoxylin eosin(HE) and IHC staining respectively.The effects of the three sectioning methods on HE and IHC staining were compared.The results showed that the quality of cryosections is significantly improved when the sections was pre-embeded with OCT for more than 6 hours prior to sectioning;the tissue structure is much more clearer and there are less ice crystal-derived cavities in modified cryosections than in conventional cryosections;the quality of modified cryosections from lung tissue is much more better than that of paraffin sections.In a word,a new cryosectioning method have been established for IHC staining,being avoid of quick freezing in liquid nitrogen,reducing difficulties in multiple tissue sectioning,and thus archiving satisfied IHC staining.The new method could simplify the sectioning procedure and would contribute to antigen detection and investigation of histomorphology.
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基本信息:
DOI：10.13417/j.gab.039.005520
中图分类号:Q95-33
引用信息:
[1]潘建洪,陈秋玉,邢青波,等.动物组织冷冻切片法的改良及应用[J].基因组学与应用生物学,2020,39(12):5520-5525.DOI:10.13417/j.gab.039.005520.
基金信息:
国家自然科学基金(31660653);; 广西自然科学基金(2018GXNSFDA281026);; 广西科技计划重点研发项目(桂科AB16380098)共同资助
2020-12-25
2020-12-25