Application of vital staining with Acridine Orange (A.O.) to investigation of embryonic growth at the stage of organogenesis

本研究采用吖啶橙子活体染色荧光显微镜观察了着床后胚胎的正常和异常生长情况。来自注射AO的母鼠的胚胎在注射后5分钟发出强烈的次级荧光，并且在24小时仍然发出弱的次级荧光。after.但是48小时。孕鼠在妊娠第9天同时给予丝裂霉素C和咖啡因。众所周知，这种治疗会导致胚胎的指畸形发生率很高。在孕10、11天，即AO注射后1小时，将胚胎从子宫角取出，用荧光显微镜观察，从时间过程的观察证实，丝裂霉素C和咖啡因处理的胚胎损伤组织细胞中的次级荧光存在时间比对照组长，由于活的胚胎细胞能分泌AO，而受损的细胞不能分泌AO，胚胎全身弥漫有大量颗粒状细胞团，呈黄绿色强荧光，在肢芽间充质组织中特异性高密度存在，无指状射线形成。利用荧光显微镜，通过AO活体染色，可以通过荧光的颜色和强度来详细地研究正常和异常胚胎的生长，结合全胚胎培养的进一步研究有望为我们带来关于正常和异常胚胎生长的丰富结果。

In this study, normal and abnormal growth of post-implantation embryos was investigated by means of vital staining with acridine orange using fluorescence microscope. Embryos from mother mice injected with AO, emitted intense secondary fluorescence five minutes after injection and still did emit weak secondary one 24 hrs. after. But 48 hrs. after, embryos did illuminate only auto-fluorescence without the secondary one.Pregnant mice were administered with Mitomycin C and Caffeine simultaneously on 9th day of gestation. This treatment has been well known to cause digital malformation of the embryo at a high incidence. Then, on the successive 10th and 11th day of gestation, one hr. after AO injection into mother mice, embryos were taken out of uterine horns and investigated by fluorescence microscope.From the time course investigation, we confirmed that the secondary fluorescence existed longer in damaged tissue cells of embryos by treatment with Mitomycin C and caffeine than in control ones, as living embryonic cell can excrete AO while damaged cells can not.There were many granule-like cell masses with intense yellowish-green fluorescence diffusely on the whole body of embryo and those specifically existed with high density at the mesenchymal tissue of limb buds without formation of digital rays.From the results, above mentioned, normal and abnormal embryonic growth can be investigated in detail by color and intensity of fluorescence using fluorescence microscopy through AO vital staining.Further investigation in combination with whole embryo culture is expected to bring us a fruitful result about normal and abnormal embryonic growth.