CN108226470A - 一种在早期胚胎组织免疫组化中保持胚胎完整性的方法 - Google Patents
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Abstract
本发明公开了一种早期胚胎组织的免疫组化中保持胚胎完整性的方法,属于动物生物技术领域。该方法采用聚乙烯吡咯烷酮溶液添加于早期胚胎免疫组化所用磷酸盐缓冲液、多聚甲醛、Triton X‑100、双氧水中,防止胚胎贴壁,在整个免疫组化过程中可保持胚胎完整性,提高免疫组化效果。本发明所采用的技术方案主要针对体外胚胎,在常规免疫组化步骤基础上进行了调整,调整了组织处理部分试剂成分,有效提高了免疫组化效果。本发明针对免疫组化技术存在的不足,扩大了应用范围,具有操作简便,生产成本低等优点。
Description
技术领域
本发明涉及一种在早期胚胎组织免疫组化中保持胚胎完整性的方法,属于动物生物技术领域。
背景技术
聚乙烯吡咯烷酮简称PVP,是N-乙烯基-2-吡咯烷酮发生聚合生产的高分子化合物。其是一种白色有吸湿性的粉末,无臭或微臭,可溶于水、乙醇、氯仿和多数有机溶剂,不溶于***,毒性较小。聚乙烯吡咯烷酮可以单体乙烯基吡咯烷酮为原料,通过本体聚合、溶液聚合等方法合成。作为一种合成水溶性高分子化合物,具有水溶性高分子化合物一般性质,胶体保护作用、成膜性、粘结性、吸湿性、增溶或凝聚作用。其溶液毒性很低、无生理形容性。在医药、食品、化妆品等领域广泛应用。
体外胚胎培养为探究早期胚胎发育提供了必要的材料,是一种生殖生物技术,其加快了畜禽遗传改良,在动物育种中有着重要意义。这一技术也是哺乳动物胚胎工程研究中的一个重要工具,其为探究体内胚胎发育机制、改良胚胎发育质量奠定了良好的基础,是体内胚胎***的一个有效的反映,也是发育生物学和新兴生物技术中一个重要的研究技术。但在培养过程中,胚胎容易贴壁,在换培养液移动胚胎时会使其囊壁破裂,所以在整个培养过程中将丢失很多胚胎,建设了试验样本。利用聚乙烯吡咯烷酮水溶性、粘性及低毒性,在胚胎培养液中添加聚乙烯吡咯烷酮,可降低胚胎贴壁,保持胚胎的完整性,基于此,在胚胎免疫组化试验中,所用试剂添加一定浓度的聚乙烯吡咯烷酮,亦可减少胚胎贴壁,保持胚胎完整性,增加试验效果。
本方法在胚胎免疫组化中的应用,建立了可行的试验方法,操作简便易行,试验效果明显,为胚胎工程研究提供了切实可行的依据。
发明内容
技术问题 本发明的目的在于提供一种在早期胚胎组织免疫组化中保持胚胎完整性的方法。
技术方案
1、一种在早期胚胎组织免疫组化中保持胚胎完整性的方法,包括以下步骤:
1)溶液配制:4%多聚甲醛,0.5% Triton X-100,双氧水20%聚乙烯吡咯烷酮储存溶液和0.1μg/mL4',6-二脒基-2-苯基吲哚均用PH范围为7.2-7.4的0.01mol/L磷酸盐缓冲液稀释;且免疫组化用4%多聚甲醛,0.5% Triton X-100、双氧水和磷酸盐缓冲液中均加入聚乙烯吡咯烷酮储存液,添加浓度分别为2%、4%、6%、8%、10%。
2)胚胎清洗:在显微镜下,将体外培育的2细胞期、4细胞期、8细胞期和16细胞期的孤雌胚胎、孤雄胚胎或体外受精胚胎从培养液中捡出,用38.5℃下预热的加入聚乙烯吡咯烷酮的0.01mol/L磷酸盐缓冲液漂洗3 次,每次5min;
3)胚胎固定:用加入聚乙烯吡咯烷酮溶液的4%的多聚甲醛在室温下固定胚胎1h,然后用预热的加入聚乙烯吡咯烷酮溶液的磷酸盐缓冲液漂洗3次,每次5min;
4)通透:用加入聚乙烯吡咯烷酮的0.5% Triton X-100室温透化30 min,然后用预热的加入聚乙烯吡咯烷酮溶液的磷酸盐缓冲液漂洗3次,每次5 min;
5)去除内源性过氧化物酶:用加入聚乙烯吡咯烷酮的0.01mol/L磷酸盐缓冲液配置新鲜的3% H2O2,室温封闭5~10min,用预热的含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3 次,每次5 min;
6)封闭:滴血清(与二抗同一来源)孵育,37℃封闭30 min;
7)一抗孵育:轻轻吸除封闭液,加按抗体说明用磷酸缓冲液稀释的一抗覆盖于胚胎中,将其放在湿盒中,4℃过夜;
8)二抗孵育:用预热的含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3 次,每次5 min,加按抗体说明用磷酸缓冲液稀释的荧光标记二抗,37℃中避光孵育30 min;
9)4',6-二脒基-2-苯基吲哚染核:二抗孵育后的胚胎用含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3次,每次5 min,取10μL 0.1μg/mL4',6-二脒基-2-苯基吲哚溶液于胚胎上,室温5min;
10)封片:用含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3次,每次5 min,95%甘油封片,盖上盖玻片;
11)荧光观察:在共聚焦显微镜下观察荧光分布。
有益效果
本发明提供了一种在早期胚胎组织免疫组化中保持胚胎完整性的方法。本发明具有以下优点:1)操作简便;2)针对性强;3)提高胚胎组织免疫组化效果。本发明方法和结果为开展与早期孤雌、孤雄或体外受精胚胎有关的生物学研究提供了新的研究技术。
说明书附图
图1为孤雌胚胎发育过程;其中图a为2-4细胞期卵裂球;图b为4-16细胞期卵裂球。
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法,所述百分含量如无特别说明均为体积百分含量。
1、实施材料
体外构建的早期孤雌胚胎,包括卵裂前、2细胞胚胎、8细胞胚胎和16细胞胚胎。
2、实施方法
1)溶液配制:4%多聚甲醛,0.5% Triton X-100,双氧水20%聚乙烯吡咯烷酮储存溶液和0.1μg/mL 4',6-二脒基-2-苯基吲哚均用PH范围为7.2-7.4的0.01mol/L磷酸盐缓冲液稀释;且免疫组化用4%多聚甲醛,0.5% Triton X-100、双氧水和磷酸盐缓冲液中均加入聚乙烯吡咯烷酮储存液,添加浓度分别为2%、4%、6%、8%、10%;
2)胚胎清洗:在显微镜下,将体外培育的2细胞期、4细胞期、8细胞期和16细胞期的孤雌胚胎、孤雄胚胎或体外受精胚胎从培养液中捡出,用38.5℃下预热的含聚乙烯吡咯烷酮的0.01mol/L磷酸盐缓冲液漂洗3 次,每次5min;
3)胚胎固定:用含聚乙烯吡咯烷酮溶液的4%的多聚甲醛在室温下固定胚胎1h,然后用预热的含聚乙烯吡咯烷酮溶液的磷酸盐缓冲液漂洗3次,每次5min;
4)通透:用含聚乙烯吡咯烷酮的0.5% Triton X-100室温透化30 min,然后用预热的含聚乙烯吡咯烷酮溶液的磷酸盐缓冲液漂洗3次,每次5 min;
5)去除内源性过氧化物酶:用含聚乙烯吡咯烷酮的0.01mol/L磷酸盐缓冲液配置新鲜的3% H2O2,室温封闭5~10min,用预热的含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3 次,每次5 min;
6)封闭:滴血清(与二抗同一来源)孵育,37℃封闭30 min;
7)一抗孵育:轻轻吸除封闭液,加1:100磷酸盐缓冲液稀释的抗MT1多克隆抗体覆盖于胚胎中,将其放在湿盒中,4℃过夜;
8)二抗孵育:用含的聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3 次,每次5 min,加1:100磷酸盐缓冲液稀释的CY3标记羊抗兔荧光二抗,37℃中避光孵育30 min;
9)4',6-二脒基-2-苯基吲哚染核:二抗孵育后的胚胎用含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3次,每次5 min,取适量0.1μg/mL 4',6-二脒基-2-苯基吲哚于胚胎上,室温5min;
10)封片:用含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3次,每次5 min,95%甘油封片,盖上盖玻片;
11)荧光观察:在共聚焦显微镜下观察荧光分布。
3、处理效果
体外胚胎在培养过程中容易贴壁,致使一些胚胎在一些试验操作过程中无法保证其完整性,而丢失许多胚胎。添加聚乙烯吡咯烷酮可以防止胚胎贴壁,利于试验中对胚胎的操作,保持胚胎完整性。表1显示,在免疫组化试验过程中,所用试剂中添加不同浓度聚乙烯吡咯烷酮溶液,胚胎完整性受到很大影响。随着添加的聚乙烯吡咯烷酮浓度增加,胚胎完整率逐渐增加,以8%聚乙烯吡咯烷酮溶液处理后的胚胎完整性是最高的,可达到95.24%;但当添加10% 聚乙烯吡咯烷酮溶液后,胚胎完整率下降到72%。由此可见,胚胎免疫组化试验中,添加8%的聚乙烯吡咯烷酮溶液,胚胎的完整率是最高的,增加了试验效率。
表1 不同浓度聚乙烯吡咯烷酮溶液对胚胎完整性的影响
聚乙烯吡咯烷酮溶液(%) | 胚胎数(个) | 完整胚胎数(个) | 完整率(%) |
2 | 20 | 9 | 45.00 |
4 | 22 | 11 | 50.00 |
6 | 24 | 18 | 75.00 |
8 | 21 | 20 | 95.24 |
10 | 25 | 18 | 72.00 |
Claims (2)
1.一种在早期胚胎组织免疫组化中保持胚胎完整性的方法,包括以下步骤:
1)溶液配制:4%多聚甲醛,0.5%Triton X-100,双氧水20%聚乙烯吡咯烷酮储存溶液和0.1μg/mL 4',6-二脒基-2-苯基吲哚均用PH范围为7.2-7.4的0.01mol/L磷酸盐缓冲液稀释,并且免疫组化用4%多聚甲醛,0.5%Triton X-100、双氧水和磷酸盐缓冲液中均加入聚乙烯吡咯烷酮储存液,添加浓度分别为2%、4%、6%、8%、10%;
2)胚胎清洗:在显微镜下,将体外培育的2细胞期、4细胞期、8细胞期和16细胞期的孤雌胚胎、孤雄胚胎或体外受精胚胎从培养液中捡出,用38.5℃下预热的加入聚乙烯吡咯烷酮的0.01mol/L磷酸盐缓冲液漂洗3次,每次5min;
3)胚胎固定:用加入聚乙烯吡咯烷酮溶液的4%的多聚甲醛在室温下固定胚胎1h,然后用预热的加入聚乙烯吡咯烷酮溶液的磷酸盐缓冲液漂洗3次,每次5min;
4)通透:用加入聚乙烯吡咯烷酮的0.5%Triton X-100室温透化30min,然后用预热的加入聚乙烯吡咯烷酮溶液的磷酸盐缓冲液漂洗3次,每次5min;
5)去除内源性过氧化物酶:用加入聚乙烯吡咯烷酮的0.01mol/L磷酸盐缓冲液配置新鲜的3%H2O2,室温封闭5-10min,用预热的含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3次,每次5min;
6)封闭:滴血清(与二抗同一来源)孵育,37℃封闭30min;
7)一抗孵育:轻轻吸除封闭液,加按抗体说明用磷酸缓冲液稀释的一抗覆盖于胚胎中,将其放在湿盒中,4℃过夜;
8)二抗孵育:用预热的含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3次,每次5min,加按抗体说明用磷酸缓冲液稀释的荧光标记二抗,37℃中避光孵育30min;
9)4',6-二脒基-2-苯基吲哚染核:二抗孵育后的胚胎用含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3次,每次5min,取10μL 0.1μg/mL 4',6-二脒基-2-苯基吲哚溶液于胚胎上,室温5min;
10)封片:用含聚乙烯吡咯烷酮的磷酸盐缓冲液漂洗3次,每次5min,95%甘油封片,盖上盖玻片;
11)荧光观察:在共聚焦显微镜下观察荧光分布。
2.根据权利要求1所述的一种在早期胚胎组织免疫组化中保持胚胎完整性的方法,其特征在于:所述聚乙烯吡咯烷酮溶液为PH范围为7.2-7.4的0.01mol/L磷酸盐缓冲液稀释溶液。
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