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技能篇 | IHC之路漫漫（下篇）

发布: 2019-01-09 672阅读 225评论

在上期：IHC之路漫漫（上篇）中，我们的实验达人对在IHC实验中，实验结果如何分析、Triton X-100的使用、如何才能充分脱蜡、一般在什么情况下进行组织抗原修复等问题进行经验总结，接下来我们将继续一抗的选择、如何减少非特异性染色等问题进行学习。

7. 封闭血清该如何选择？

实验中使用封闭血清，以消除非特异性染色，提高目的蛋白的准确性，同时降低背景。

封闭血清一般是和二抗同一来源的，由于二抗大多数是山羊二抗，因此常用山羊血清。有时也会用小牛血清、BSA、羊血清等，但不能与一抗的来源相同。

8. 一抗选择要点

一抗的选择是IHC实验能否成功的关键：

（1）单克隆或多克隆抗体的选择，各有优缺，根据实验目的决定。

一般单克隆抗体特异性强，但亲和力相对小，检测抗原灵敏度相对就低；而多克隆抗体特异性稍弱，但抗体的亲和力强，灵敏度高。

（2）确定一抗的应用范围

根据自己切片状况（石蜡或冰冻），选择适用IHC的抗体。

（3）一抗的种属来源决定选择相应的二抗

有的抗体可能存在种属差异。

（4）生产厂家的选择

尽量选择可以提供技术支持的制造商，这样可以详细的了解一抗的种属、来源、抗原、质检等信息。

9. 一抗在4℃孵育后为什么要进行37℃复温？

（1）防止脱片

切片如果从4℃取出后直接放入PBS缓冲液中，易发生脱片。

（2）使抗原抗体结合更稳定

对于一些表达较弱的抗原来说，这一步是必需的，在4℃时，抗原抗体分子碰撞几率和运动速率较低，在37℃时，抗原抗体结合更快。但同时也会提高敏感性，造成非特异性染色。

10. 脱片产生的原因以及如何防止脱片？

（1）黏附载玻片质量的问题

（2）组织切的不好，或切片机的问题——如比较钝的刀片切的厚或不均匀、切片者手法不好等

（3）组织问题，比如癌症组织有坏死等

（4）没烤好，时间短或温度不够

（5）操作时甩的太剧烈，有脱片嫌疑的片子最好不甩或轻甩，可用纸从边缘吸水

（6）修复问题：抗原修复时高压时间过长，或者放进修复液时直接丢入等。此外，用EDTA修复比柠檬酸更易脱片。

（7）当见到有组织漂起来时操作更要谨慎，用PBS时尽量用泡的。

11. DAB显色时间如何把握？

（1）DAB显色时间不固定的，主要在显微镜下控制显色时间，到出现阳性部分深棕色，无背景时即可冲洗；

（2）若DAB显色时间很短（如几秒或几十秒）就出现很深的棕褐色，可能意味着你的抗体浓度过高或抗体孵育时间过长，需要下调抗体浓度或缩短抗体孵育时间；

（3）若短时间就出现很深背景，可能是前面封闭非特异性蛋白不完全，需要延长封闭时间；

（4）DAB显色时间很长（如超过十几分钟）才出现阳性染色，一方面可能说明抗体浓度过低或孵育时间过短（最好一抗4度过夜）；另一方面就是封闭时间过长。

12. 造成切片染色深的因素有哪些？如何才能减少非特异性染色？

（1）抗体浓度过——一抗浓度过高是常见的原因之一

解决措施：每次使用新抗体前应当对其工作浓度进行测试，使每一抗体个体化，找到适合自己实验室的理想工作浓度，既使是即用型的抗体也应如此，不能只简单的按说明书进行染色。

（2）抗体孵育时间过长或温度较高

解决措施：严格执行操作规程，避免因遗忘而造成时间延长。现在流行的二步法（Polymer）敏感性很高，要求一抗孵育的时间不是传统的1H，而是30min，因此，要根据染色结果进行调整。

（3）DAB变质和显色时间太长

解决措施：DAB最好现用现配，如有沉渣，过滤后再用。

配制好的DAB不能长时间存放，因为在没有酶的情况下，过氧化氢也会游离出氧原子与DAB产生反应而降低其效力，因此未用完的DAB存放在冰箱几天后再用也是不可取的。DAB的显色最好在显微镜下监控，达到理想染色程度时立即终止反应。

（4）组织变干——修复液溢出后未及时补充液体、染色切片太多、动作太慢、忘记滴液、滴液流失等原因造成。

解决措施：操作时要认真仔细，采用DAKO笔或PAP Pen在组织周围画圈，可以有效的避免液体流失，也能提高操作速度。

（5）一抗变质、质量差的多克隆抗体

解决措施：注意抗体有效期，过期抗体会出现不显色或背景着色。

好的实验结果，是发一篇好Paper的重要前提。希望以上经验分享能让大家获得一个好的前提。

篇幅有限，IHC实验过程中肯定还有更多的问题，欢迎大家在文章底部留下你在实验中遇到的难题，我们会邀请有着多年IHC实验经验的大佬们来为大家解答。

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