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免疫熒光技術(shù)是將免疫學(xué)方法(抗原抗體特異結(jié)合)與熒光標(biāo)記技術(shù)結(jié)合起來(lái)研究特異蛋白抗原在細(xì)胞內(nèi)分布的方法。由于熒光素所發(fā)的熒光可在熒光顯微鏡下檢出，從而可對(duì)抗原進(jìn)行細(xì)胞定位。 免疫熒光細(xì)胞化學(xué)是根據(jù)抗原抗體反應(yīng)的原理，先將已知的抗原或抗體標(biāo)記上熒光素制成熒光標(biāo)記物，再用這種熒光抗體（或抗原）作為分子探針檢查細(xì)胞或組織內(nèi)的相應(yīng)抗原（或抗體）。在細(xì)胞或組織中形成的抗原抗體復(fù)合物上含有熒光素，利用熒光顯微鏡觀察標(biāo)本，熒光素受激發(fā)光的照射而發(fā)出明亮的熒光，可以看見(jiàn)熒光所在的細(xì)胞或組織，從而對(duì)抗原或抗體進(jìn)行定性、定位或定量檢測(cè)。

免疫熒光檢測(cè)方法根據(jù)待檢測(cè)抗原的豐度采用不同的熒光標(biāo)記策略。對(duì)于高豐度的抗原，可采用熒光標(biāo)記一抗直接檢測(cè)的方式；對(duì)于中等豐度的抗原，可采用熒光標(biāo)記二抗的方式對(duì)待檢測(cè)的抗原進(jìn)行信號(hào)放大；對(duì)于低等豐度的抗原，可采用生物素標(biāo)記抗體和熒光標(biāo)記鏈霉親和素的方式對(duì)待檢測(cè)的抗原進(jìn)行二級(jí)信號(hào)放大。在整個(gè)免疫熒光檢測(cè)的過(guò)程中，熒光染料的性能（信號(hào)強(qiáng)度，穩(wěn)定性，背景信號(hào)）對(duì)免疫熒光檢測(cè)的結(jié)果發(fā)揮著關(guān)鍵性的作用。早期較常用的FITC熒光素標(biāo)記抗體由于其信號(hào)弱，穩(wěn)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(yōu)良的Andy Fluor熒光染料。這些新型熒光染料跟傳統(tǒng)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(biāo)記的二抗及相應(yīng)的標(biāo)記服務(wù)，為廣大科研用戶提供了廣闊的選擇。

熒光標(biāo)記二抗及鏈霉親和素選購(gòu)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).