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目的:探讨微小组织Histo Gel预包埋的石蜡切片制作方法和优势。方法:选择拟胚体(embryoid body,EB)为微小组织,将人类胚胎干细胞(human Embryonic Stem Cell,h ESC)切割成小块,用拟胚体培养基在低贴附培养皿中悬浮培养形成拟胚体。收集拟胚体,4%的多聚甲醛固定。将Histo Gel加热到60℃熔解,离心去除拟胚体中的固定液,把液态的Histo Gel加到拟胚体上,调整拟胚体的相对位置使其相对集中,待胶冷却凝固,将含有拟胚体的胶块取出,进行常规的石蜡切片操作,包括脱水、透明、浸蜡、包埋、切片和苏木素-伊红(hematoxylin-eosin,HE)染色。显微镜下观察拟胚体的形态,并用拟胚体的形态完好度来评判这种方法。结果:Histo Gel仅在60℃便可熔解,室温可以冷却凝固,含有拟胚体的胶块在整个操作过程中很方便。展片过程中,石蜡和Histo Gel能够保持平整。HE染色的结果可以看出,拟胚体内部结构完好,细胞核和细胞质清晰可辨。结论:Histo Gel可以作为一种微量细胞组织的预包埋胶制作石蜡切片,而且相比琼脂预包埋,Histo Gel因其操作更加方便和特有的物理特性显示出更多的优点。
OBJECTIVE: To investigate the method and advantage of making paraffin sections embedded in Histo Gel micro-tissue. Methods: Human embryonic stem cells (EBs) were selected as microstructures. Human embryonic stem cells (hESCs) were cut into small pieces and cultured in embryos with low attachment petri dishes Embryoid body. Embryoid body was collected and fixed with 4% paraformaldehyde. The Histo Gel is heated to 60 ° C. to melt, centrifuged to remove the fixing solution in the embryoid body, the liquid Histo Gel is added to the embryoid body, the relative position of the embryoid body is adjusted to be relatively concentrated, the gel is cooled and solidified, The embryoid body gel was removed and subjected to conventional paraffin sections, including dehydration, clearing, dipping, embedding, sectioning and hematoxylin-eosin (HE) staining. The morphology of the embryoid body was observed under a microscope, and the method was judged by the morphology integrity of the embryoid body. Results: Histo Gel melted at 60 ° C, allowed to cool and solidify at room temperature, and the gel-like mass containing the embryoid body was convenient for the entire operation. Paraffin and Histo Gel remained flat during the exhibition. The result of HE staining shows that the internal structure of embryoid body is intact and the nucleus and cytoplasm are clear and distinct. CONCLUSIONS: Histo Gel can be used as a paraffin gel for the preparation of a micro-cellular pre-encapsulated gel, and Histo Gel shows more advantages due to its easier handling and unique physical properties than agar pre-embedding.