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免疫熒光技術是將免疫學方法(抗原抗體特異結合)與熒光標記技術結合起來研究特異蛋白抗原在細胞內分布的方法。由于熒光素所發的熒光可在熒光顯微鏡下檢出，從而可對抗原進行細胞定位。 免疫熒光細胞化學是根據抗原抗體反應的原理，先將已知的抗原或抗體標記上熒光素制成熒光標記物，再用這種熒光抗體（或抗原）作為分子探針檢查細胞或組織內的相應抗原（或抗體）。在細胞或組織中形成的抗原抗體復合物上含有熒光素，利用熒光顯微鏡觀察標本，熒光素受激發光的照射而發出明亮的熒光，可以看見熒光所在的細胞或組織，從而對抗原或抗體進行定性、定位或定量檢測。

免疫熒光檢測方法根據待檢測抗原的豐度采用不同的熒光標記策略。對于高豐度的抗原，可采用熒光標記一抗直接檢測的方式；對于中等豐度的抗原，可采用熒光標記二抗的方式對待檢測的抗原進行信號放大；對于低等豐度的抗原，可采用生物素標記抗體和熒光標記鏈霉親和素的方式對待檢測的抗原進行二級信號放大。在整個免疫熒光檢測的過程中，熒光染料的性能（信號強度，穩定性，背景信號）對免疫熒光檢測的結果發揮著關鍵性的作用。早期較常用的FITC熒光素標記抗體由于其信號弱，穩定性差，目前正逐漸被新型熒光染料（如Alexa Fluor, Dylight）所取代。

Applied BioProbes公司推出了一系列性能優良的Andy Fluor熒光染料。這些新型熒光染料跟傳統的熒光染料（如FITC）相比，具有*的*性。公司新推出了一系列Andy Fluor和生物素標記的二抗及相應的標記服務，為廣大科研用戶提供了廣闊的選擇。

熒光標記二抗及鏈霉親和素選購指南

Andy Fluor™ dye conjugates have brighter fluorescence than other fluorophores.

Figure 1. Comparison of relative fluorescence of goat anti-rabbit IgG antibody conjugates prepared from Andy Fluor™ 488, 555, and 647 with Cy®2, Cy®3, and Cy®5.

Figure 2. Flow cytometry comparison of the brightness of goat anti-mouse IgG antibody conjugates prepared from Andy Fluor™ 488, 555, and 647 with Cy®2, DyLight™ 488, Cy®3, DyLight™ 550, and DyLight™ 650.

Andy Fluor™ dye conjugates have better photostability than other fluorophores.

Figure 3. Comparison of the photobleaching rates of Andy Fluor™ 488 goat anti-mouse IgG (H+L) (L109B) with FITC goat anti-mouse IgG (H+L) (L146B). The cytoskeleton of HeLa cells was labeled with mouse monoclonal anti-α-tubulin antibody in combination with Andy Fluor™ 488 goat anti-mouse IgG (H+L) antibody (top series) or with mouse monoclonal anti-α-tubulin antibody in combination with FITC goat anti-mouse IgG (H+L) antibody (bottom series). The fluorescence imaging was taken at 60-second intervals (0, 60, and 120 seconds of exposure).

Figure 4. Immunofluorescent stain of α-tubulin in BEAS2BNNK cells. α-Tubulin in fixed and permeabilized BEAS2BNNK cells was labeled with anti-α-tubulin primary antibody, and then visualized with goat anti-mouse IgG antibodies conjugated with either Andy Fluor™ 488 (top, left), Andy Fluor™ 555 (top, right), Andy Fluor™ 568 (bottom, left), or Andy Fluor™ 647 (bottom, right). Nuclei are counterstained with DAPI (blue).

Figure 5. Immunofluorescent stain of CCSP in mouse lung tissue. FFPE samples of mouse lung were labeled with rabbit anti-CCSP primary antibody, and then visualized with green-fluorescent Andy Fluor™ 488 goat anti-rabbit IgG antibody (green). Nuclei were counterstained with DAPI (blue).

Figure 6. Immunofluorescent stain of CCSP in mouse lung tissue. FFPE samples of mouse lung were labeled with rabbit anti-CCSP primary antibody, and then visualized with either Andy Fluor™ 555 (red, left), or Andy Fluor™ 568 (red, right). Nuclei were counterstained with DAPI (blue).

Figure 7. Immunofluorescent stain of α-tubulin in BEAS2BNNK cells and CCSP in mouse lung tissue. α-Tubulin in fixed and permeabilized BEAS2BNNK cells was labeled with anti-α-tubulin primary antibody, followed by incubation with biotin goat anti-mouse IgG antibody, and then visualized with Andy Fluor™ 488 Streptavidin (green, left). FFPE samples of mouse lung were labeled with rabbit anti-CCSP primary antibody, followed by incubation with biotin goat anti-mouse IgG antibody, and then visualized with Andy Fluor™ 488 Streptavidin (green, middle and right). Nuclei were counterstained with DAPI (blue).