免疫熒光（IF）染色及最佳化技巧
什麼是IF（免疫熒光）染色？
What is IF （Immunofluorescence） staining？
免疫熒光（IF）是一種用於檢測以及識別細胞和組織中蛋白質和其他分子的亞細胞分佈和遷移的強大檢測技術。IF可用作免疫組織化學 （IHC） 或免疫細胞化學 （ICC） 中使用的傳統顯色標記的替代物。透過使用多種標記物的二級抗體，可以研究感興趣蛋白的共定位，這是不能透過常規IHC或ICC實現。這是同時分析許多目標蛋白時的一個優勢，可同時生成大量亞細胞定位的準確資料。
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。
什麼時候使用IF？
When to use IF？
● IF是一個很好的檢測技術工具，用於研究感興趣的產物如分子、蛋白質或糖蛋白的表達、定位、分佈和遷移。
IF is an excellent tool to illustrate expression， localization， distribution and movement of products of interest such as molecules， proteins or glycoproteins。
● 用作顯色標記物的替代物，例如3，3 ‘-二氨基聯苯胺 （DAB） 。
IF is used as an alternative to chromogenic labelings， such as 3，3’-Diaminobenzidine （DAB）。
IF與ICC和IHC的區別
The difference between IF & ICC & IHC
IHC和ICC使用的樣本型別不同。IHC利用通常冷凍或石蠟包埋的整個組織樣本。ICC指的是分離的或培養的細胞樣本。這兩種樣本型別都可以使用IF進行標記檢測。直接法IF使用帶有偶聯熒光染料（共軛抗體）的一級抗體。間接法IF需同時使用可結合靶標抗原一抗和偶聯熒光染色的二抗。
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&；amp；amp；間接法
IF問題疑難解答
Troubleshooting IF Issues
實驗問題Issues
解決辦法 Solutions
非特異性染色Non-Specific Staining
●檢查所用熒光團的光譜是否重疊：調整濾鏡和光源，更改為激發光譜或發射光譜沒有重疊的熒光團。Check for spectral overlap of fluorophores used。 Adjust filters and light sources。 Change to fluorophores which don’t have overlapping excitation or emission spectra。
●一抗種屬來源與被分析的樣本物種相同：更換抗體；使用與二抗來源物種相同的血清作為封閉液；用針對一抗種屬的單價Fab片段封閉一抗，然後用二抗對單價Fab片段進行視覺化，可避免與樣本本身的Fc受體結合而引起非特異染色。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。
●切片中存在雜質引起非特異熒光：二抗使用前離心，將不溶雜質或遊離的染料沉澱到底部，吸取上清進行實驗；甲醛固定後樣本，可用甘氨酸淬滅殘留的醛。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
●可能是由多種原因引起的：檢查曝光時間和強度；改變固定或孵育時間；確保抗體/同型的相容性；測試抗體稀釋範圍和樣品濃度；保證正確的試劑和載玻片的製備和儲存方法；檢查裝置不相容性。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。
背景訊號Background Signal
●試劑或樣本問題：降低抗體濃度（一級或二級）；使用較薄的組織切片；透過對照檢查二抗特異性，如果發生非特異性結合，則更換二抗。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。
●封閉可能不足：增加封閉孵育時間或使用其他封閉劑；用與二抗種屬相同物種的正常血清封閉。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。
●可能是由於自發熒光引起的：檢查未染色的部分；遊離醛可以用硼氫化鈉洗滌除去，並避免使用戊二醛固定劑；預光漂白內源性分子，導致使用蘇丹黑染色；減少顯色的孵育時間；考慮遠離488 nm通道，並使用與自發熒光不同的發射波長的熒光團。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。
最佳化IF
Optimizing IF
以下IF實驗最佳化小技巧可以節省您的時間和成本，從而避免浪費您的寶貴樣品。
These tips and tricks will save you both time and costs by optimizing each step， which will prevent wasting your valuable samples。
1。 設定對照：可以使用陽性和陰性對照來確定結果的準確性，並幫助確定任何問題的來源。
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。 樣品和試劑的儲存：由於IF中所用抗體和實驗樣品的熒光性質，因此必須適當儲存。最好將熒光抗體和經過IF處理的樣品都儲存在完全黑暗的環境中。使用琥珀色或鋁箔覆蓋抗體管。
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。 使用適當的固定劑：醛可用於標記膜結合抗原或細胞骨架抗原，並應用於核蛋白或線粒體蛋白。醛固定後的樣本需要抗原修復和打孔滲透步驟才能正確檢測抗原。如果使用單克隆抗體推薦有機溶劑用於組織固定。甲醇適用於冷凍樣本的固定；丙酮固定對組織學儲存效果較好。
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。 仔細選擇抗體：應檢查所有抗體，無論是單克隆抗體還是多克隆抗體，以確保與目標抗原具有高的親和力。所用的二抗必須與一抗相容，比如使用抗小鼠二抗檢測小鼠來源的一抗。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：最好使用在不同物種中產生的一抗。如果使用間接檢測，請使用已預先吸附在檢測其他蛋白質的一抗和二抗的種屬以及實驗樣品的物種中的二抗。這樣可以最大程度地減少跨物種的反應性和非特異性結合。所有二抗也應在同一物種中產生，以最大程度地減少交叉反應。一旦確定抗體特異性，就可以同時或依次將目標抗原與其抗體孵育，順序孵育往往會產生更好的結果。對於亞細胞靶標的共定位或復染色，請選擇對細胞器標記特異的抗體。
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。 決定直接或間接染色：兩種方法都有優點和缺點，根據實驗需求選擇。使用這兩種方法，都應該確保充足的洗滌以最小化非特異性的結合。
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。 選擇沒有光譜重疊的熒光團：IF是一種很好的多路複用分析技術，測量如此多的引數，那麼使用能夠提供可分辨訊號的熒光團是很有必要的。
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。
