免疫荧光（IF）染色及优化技巧

营养师

什么是IF（免疫荧光）染色？What is IF (Immunofluorescence) staining?
免疫荧光（IF）是一种用于检测以及识别细胞和组织中蛋白质和其他分子的亚细胞分布和迁移的强大检测技术。IF可用作免疫组织化学 (IHC) 或免疫细胞化学 (ICC) 中使用的传统显色标记的替代物。通过使用多种标记物的二级抗体，可以研究感兴趣蛋白的共定位，这是不能通过常规IHC或ICC实现。这是同时分析许多目标蛋白时的一个优势，可同时生成大量亚细胞定位的准确数据。
Immunofluorescence (IF) is a powerful tool used to detect and identify the subcellular distribution and movement of proteins and other molecules within cells and tissues. IF can be used as an alternative to traditional chromogenic labeling used in Immunohistochemistry (IHC) or Immunocytochemistry (ICC). By using multiple secondary antibodies, the colocalization of proteins of interest can be studied, which cannot be achieved through conventional IHC or ICC. This is an advantage when analyzing many targets at the same time, generating a high volume of accurate data. Please see our [Immunofluorescence protocol] for a step-by-step guide.
什么时候使用IF？When to use IF？
● IF是一个很好的检测技术工具，用于研究感兴趣的产物如分子、蛋白质或糖蛋白的表达、定位、分布和迁移。
IF is an excellent tool to illustrate expression, localization, distribution and movement of products of interest such as molecules, proteins or glycoproteins.
● 用作显色标记物的替代物，例如3,3 '-二氨基联苯胺 (DAB) 。
IF is used as an alternative to chromogenic labelings, such as 3,3'-Diaminobenzidine (DAB).
IF与ICC和IHC的区别The difference between IF & ICC & IHC
IHC和ICC使用的样本类型不同。IHC利用通常冷冻或石蜡包埋的整个组织样本。ICC指的是分离的或培养的细胞样本。这两种样本类型都可以使用IF进行标记检测。直接法IF使用带有偶联荧光染料（共轭抗体）的一级抗体。间接法IF需同时使用可结合靶标抗原一抗和偶联荧光染色的二抗。
IHC and ICC differ in the type of sample used. IHC utilizes whole tissue samples which are usually frozen or paraffin-embedded. ICC refers to either isolated or cultured cells instead. Both methods can use IF as a labeling mechanism. Direct IF uses a primary antibody with an attached fluorescent dye (a conjugated antibody). Indirect IF uses a primary antibody that binds to the target and a secondary conjugated antibody that binds to the primary.



IF的直接法&间接法

IF问题疑难解答 Troubleshooting IF Issues

实验问题Issues	解决办法  Solutions
非特异性染色Non-Specific Staining	●检查所用荧光团的光谱是否重叠：调整滤镜和光源，更改为激发光谱或发射光谱没有重叠的荧光团。Check for spectral overlap of fluorophores used. Adjust filters and light sources. Change to fluorophores which don’t have overlapping excitation or emission spectra. ●一抗种属来源与被分析的样本物种相同：更换抗体；使用与二抗来源物种相同的血清作为封闭液；用针对一抗种属的单价Fab片段封闭一抗，然后用二抗对单价Fab片段进行可视化，可避免与样本本身的Fc受体结合而引起非特异染色。Primary antibody raised against the same species as tissue being analyzed：Change antibodies；Use normal serum from the host species of the secondary antibody as a blocking serum；Block primary antibody with monovalent Fab fragments raised against the primary antibody host species and then use secondary antibody to the host species of the monovalent Fab fragments to visualize. ●切片中存在杂质引起非特异荧光：二抗使用前离心，将不溶杂质或游离的染料沉淀到底部，吸取上清进行实验；甲醛固定后样本，可用甘氨酸淬灭残留的醛。Aggregates present in the sample. Microcentrifuge secondary antibodies to send aggregates to the base of the tube. After formaldehyde fixation, quench residual aldehydes with glycine.
弱染色Weak Staining	●可能是由多种原因引起的：检查曝光时间和强度；改变固定或孵育时间；确保抗体/同型的兼容性；测试抗体稀释范围和样品浓度；保证正确的试剂和载玻片的制备和储存方法；检查设备不兼容性。Can be caused by a variety of reasons：Check exposure time and intensity. Alter fixation or incubation length. Ensure antibody/ isotype compatibility. Test Ab dilution range and sample concentration. Maintain proper reagent and slide preparation and storage. Check for equipment Incompatibility.
背景信号Background Signal	●试剂或样本问题：降低抗体浓度（一级或二级）；使用较薄的组织切片；通过对照检查二抗特异性，如果发生非特异性结合，则更换二抗。Reagent or Sample issue：Reduce antibody concentration (Primary or Secondary)；Use thinner tissue sections；Check secondary binding specificity via control, change the secondary if non-specific binding occurs. ●封闭可能不足：增加封闭孵育时间或使用其他封闭剂；用与二抗种属相同物种的正常血清封闭。Blocking may be insufficient. Increase blocking incubation time or use a different blocking agent. Block with 4% normal serum from the same species as the host species for your secondary antibody. ●可能是由于自发荧光引起的：检查未染色的部分；游离醛可以用硼氢化钠洗涤除去，并避免使用戊二醛固定剂；预光漂白内源性分子，导致使用苏丹黑染色；减少显色的孵育时间；考虑远离488 nm通道，并使用与自发荧光不同的发射波长的荧光团。Might be caused by autofluorescence. Check an unstained section. Free aldehydes can be removed with a sodium borohydride wash and avoid using glutaraldehyde fixative. Pre-photobleach endogenous molecules, causing staining using Sudan black. Reduce the incubation time during amplification. Consider moving away from the 488 nm channel and working with a fluorophore emitting at a different wavelength from the autofluorescence.

优化IF Optimizing IF
以下IF实验优化小技巧可以节省您的时间和成本，从而避免浪费您的宝贵样品。
These tips and tricks will save you both time and costs by optimizing each step, which will prevent wasting your valuable samples.

1. 设置对照：可以使用阳性和阴性对照来确定结果的准确性，并帮助确定任何问题的来源。
Include controls: Both positive and negative controls can be used to ascertain the accuracy of your results and help to work out where any issues may originate from.

2. 样品和试剂的存储：由于IF中所用抗体和实验样品的荧光性质，因此必须适当存储。最好将荧光抗体和经过IF处理的样品都保存在完全黑暗的环境中。使用琥珀色或铝箔覆盖抗体管。
Sample and reagent storage: Due to the fluorescent nature of the antibodies used and the samples produced for IF, it is essential to store both appropriately. It’s best to store both the fluorescent antibodies and the IF-treated samples in complete darkness. Use amber colored or foil covered vials for the antibodies.

3. 使用适当的固定剂：醛可用于标记膜结合抗原或细胞骨架抗原，并应用于核蛋白或线粒体蛋白。醛固定后的样本需要抗原修复和打孔渗透步骤才能正确检测抗原。如果使用单克隆抗体推荐有机溶剂用于组织固定。甲醇适用于冷冻样本的固定；丙酮固定对组织学保存效果较好。
Use an appropriate fixative: Aldehydes can be used for labeling of membrane-bound or cytoskeletal antigens and should be used for nuclear or mitochondrial proteins. Aldehyde fixation requires a permeabilization step for antigen detection to work correctly. Organic solvents are recommended for monoclonal antibody use. Methanol is excellent for frozen samples; Acetone is better for histological preservation.

4. 仔细选择抗体：应检查所有抗体，无论是单克隆抗体还是多克隆抗体，以确保与目标抗原具有高的亲和力。所用的二抗必须与一抗兼容，比如使用抗小鼠二抗检测小鼠来源的一抗。Choose antibodies carefully: All antibodies, whether monoclonal or polyclonal, should be checked to ensure a high affinity with the antigen of interest. The secondary antibody used must be compatible with the primary, i.e. detect a mouse primary antibody using an anti-mouse secondary.

5. 多色染色：最好使用在不同物种中产生的一抗。如果使用间接检测，请使用已预先吸附在检测其他蛋白质的一抗和二抗的种属以及实验样品的物种中的二抗。这样可以最大程度地减少跨物种的反应性和非特异性结合。所有二抗也应在同一物种中产生，以最大程度地减少交叉反应。一旦确定抗体特异性，就可以同时或依次将目标抗原与其抗体孵育，顺序孵育往往会产生更好的结果。对于亚细胞靶标的共定位或复染色，请选择对细胞器标记特异的抗体。
Multi-color staining: It's best to use primary antibodies raised in different species. If using indirect detection, use secondary antibodies that have been pre-adsorbed against the host species of the primary and secondary antibodies detecting other proteins, as well as the species of the experimental sample. This minimizes cross-species reactivity and non-specific binding. All secondary antibodies should also be raised in the same host species to minimize cross-reactivity. Once specificity has been established, the antigens of interest can be incubated with their antibodies simultaneously or sequentially. Sequential incubations tend to generate better results. For colocalization or counterstaining of subcellular targets, choose antibodies specific to the organelle marker.

6. 决定直接或间接染色：两种方法都有优点和缺点，根据实验需求选择。使用这两种方法，都应该确保充足的洗涤以最小化非特异性的结合。
Decide on direct or indirect staining: Both methods have advantages and disadvantages. With both methods, make sure to perform a thorough washing to minimize non-specific binding.

7. 选择没有光谱重叠的荧光团：IF是一种很好的多路复用分析技术，测量如此多的参数，那么使用能够提供可分辨信号的荧光团是很有必要的。
Choose fluorophores with no spectral overlap: IF is an excellent technique for multiplexing, with so many parameters being measured, it is essential to use fluorophores that will provide discernable signals.

原文地址：https://zhuanlan.zhihu.com/p/423916349