Paragas V B, Zhang Y Z, Haugland R P, Singer V L
Molecular Probes, Inc., Eugene, OR 97402, USA.
J Histochem Cytochem. 1997 Mar;45(3):345-57. doi: 10.1177/002215549704500302.
We used the ELF-97 (Enzyme-Labeled Fluorescence) phosphatase substrate, 2-(5'-chloro-2-phosphoryloxyphenyl)-6-chloro-4(3H)-quinazolinone, with alkaline phosphatase conjugates of streptavidin and appropriate antibodies to amplify signals from biotinylated and haptenylated hybridization probes. The dephosphorylated product, ELF-97 alcohol, is a bright yellow-green fluorescent precipitate optimally excited at approximately 360 nm, with emission centered at approximately 530 nm. This large Stokes shift allows ELF-97 signals to be easily distinguished from sample autofluorescence and signals arising from counterstains or other fluorophores. The ELF-97 precipitate was extremely photostable compared to fluorescein, allowing multiple photographic exposures of samples without significant signal intensity loss. For RNA in situ hybridization, labeling was specific and localized well to targets in cultured cells, tissue sections, and whole-mount zebrafish embryos. ELF-97 signals developed in seconds to minutes and were easily distinguished from pigmented tissues or cells, unlike those obtained using colorimetric substrates. We used the substrate with singly biotinylated short oligonucleotides to detect actin mRNA in MDCK cells and actin and beta-galactosidase mRNA in LacZ+ mouse fibroblasts. We also used a biotinylated cDNA, complementary to the mRNA encoded by the constant region of the T-cell receptor beta-chain, to specifically identify T-cells in mouse lymph node tissue sections. With digoxigenin-labeled probes, we detected several developmentally expressed mRNAs in whole-mount zebrafish embryos. Hybridization to centromere repeat regions in human metaphase and interphase chromosomes was also detected; ELF-97 signals were manyfold brighter than signals obtained with fluorescein conjugates. Finally, Southern blot hybridization using singly labeled oligonucleotide probes yielded a sensitivity similar to that obtained with radioactivity.
我们使用ELF-97（酶标荧光）磷酸酶底物2-(5'-氯-2-磷酸氧基苯基)-6-氯-4(3H)-喹唑啉酮，与链霉亲和素和适当抗体的碱性磷酸酶偶联物一起，以放大来自生物素化和半抗原化杂交探针的信号。去磷酸化产物ELF-97醇是一种亮黄绿色荧光沉淀物，在约360nm处激发最佳，发射峰位于约530nm处。这种大的斯托克斯位移使得ELF-97信号能够很容易地与样品自发荧光以及由复染剂或其他荧光团产生的信号区分开来。与荧光素相比，ELF-97沉淀物具有极高的光稳定性，允许对样品进行多次拍照曝光而不会有明显的信号强度损失。对于RNA原位杂交，标记具有特异性，并且在培养细胞、组织切片和斑马鱼全胚胎中能很好地定位到靶标。ELF-97信号在数秒到数分钟内即可显现，并且与色素沉着的组织或细胞很容易区分，这与使用比色底物获得的信号不同。我们使用该底物与单生物素化的短寡核苷酸来检测MDCK细胞中的肌动蛋白mRNA以及LacZ+小鼠成纤维细胞中的肌动蛋白和β-半乳糖苷酶mRNA。我们还使用了与T细胞受体β链恒定区编码的mRNA互补的生物素化cDNA，以特异性鉴定小鼠淋巴结组织切片中的T细胞。使用地高辛标记的探针，我们在斑马鱼全胚胎中检测到了几种发育过程中表达的mRNA。还检测到了与人中期和间期染色体着丝粒重复区域的杂交；ELF-97信号比用荧光素偶联物获得的信号亮很多倍。最后，使用单标记寡核苷酸探针进行的Southern印迹杂交产生的灵敏度与放射性检测相似。
