2The University of Oklahoma, Norman, OK, USA
In situ hybridization techniques typically employ chromogenic staining by enzymatic amplification to detect domains of gene expression. We demonstrate the previously unreported near infrared (NIR) fluorescence of the dark purple stain formed from the commonly used chromogens, nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). The solid reaction product has significant fluorescence that enables the use of confocal microscopy to generate high-resolution three-dimensional (3-D) imaging of gene expression.
In situ hybridization techniques using labeled nucleic acid probes have revolutionized our ability to visualize the presence and distribution of RNA transcripts within fixed tissues and cells (1,2,. Early approaches employed radiolabeled probes, but more recent nonradioactive methods for detecting mRNA expression patterns involve both chromogenic stains and fluorescent dyes. The typical chromogenic method of detecting mRNA expression involves the incorporation of the plant steroid digoxigenin (DIG) into an RNA probe (3,4,. After annealing with the endogenous RNA, the labeled probe is detected by immunolabeling with an antibody to DIG coupled to the alkaline phosphatase (AP) enzyme. The conventional chromogenic method for detecting AP activity is a combination of nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) called NBT/BCIP (5).
In this method, the hydrolysis of BCIP by AP results in the indoxyl that dimerizes to form the leucoindigo, which is in turn oxidized by NBT to the insoluble blue 5,5′-dibromo-4,4′-dichloro indigo (BCI). In this reaction, NBT is reduced to the insoluble purple diformazan (DF), making NBT-DF (6,7). Thus, this reaction produces an insoluble dark purple precipitate that is a mixture of NBT-DF and BCI. This chromogenic method is very sensitive due to enzymatic amplification, because one AP can hydrolyze many BCIP molecules. The result is a stain that can be visualized easily by light microscopy, but it is often difficult to determine the three-dimensional (3-D) pattern of expression without advanced imaging tools such as optical projection microscopy (8).
Fluorescence methods offer the advantage of being able to determine RNA expression in three dimensions when combined with optical sectioning techniques such as confocal laser scanning microscopy (CLSM). This added depth discrimination yields greater resolution of transcripts and the ability to place RNA expression into the context of the surrounding tissues and structures. In recent years, efforts have advanced toward refining fluorescent in situ hybridization techniques in two avenues: (i) to develop approaches that generate more signal per transcript to make up for the significant loss of signal by conventional immunofluorescence approaches; and (ii) to refine multiplex approaches that permit the detection of multiple RNAs within a single sample (9).
In systems where both developments have been refined, fluorescence approaches have facilitated the identification of molecular signatures in individual cells; however, they are difficult to optimize and have a number of limitations, including high background, low signal-to-noise ratio, and instability of the signal once staining is completed. For these reasons, the standard chromogenic methods for visualizing the presence of mRNAs remain dominant. Here, we report that the advantages of fluorescence in situ hybridization are easily available, as the widely used NBT/BCIP chromogenic stain is a fluorophore, a fact that had previously gone unrecognized.Materials and Methods
Two imaging systems were used for the excitation and detection of the NBT/BCIP stain. The first, a Zeiss LSM 510 laser scanning confocal microscope with a 633-nm helium-neon (HeNe) laser and a 650-nm long pass emission filter, was used for detection of the NBT/BCIP signal. The long pass filter collects all emitted light above 650 nm. The second was a Zeiss Axio Imager Z1 system with an Hg arc lamp source, a 645-685 nm excitation band pass filter, and a 760-nm long pass emission filter (Chroma Technology; www.chroma.com) coupled to an Ocean Optics HR2000 charged-couple device (CCD) spectrometer via a 200-µm core optical fiber mounted at the focal plane of the camera port that was used to determine the emission spectra. The optical fiber only collects the light falling on its core, which for the Zeiss EC Plan Neofluar® 40×/1.3 numerical aperture (NA) objective used in this work defines a 5-µm diameter area of the sample. Spatial correlation between the image and the spectra (fiber core) was established using an x-y translator in the optical fiber mount to align it to the eyepiece crosshair reticle.
The NBT/BCIP staining protocol was performed as follows: zebrafish and lamprey whole-mount embryos incubated with DIG-labeled riboprobes were immunolabeled with a sheep anti-DIG antibody conjugated to AP (1:3000; Roche Diagnostics, Indianapolis, IN, USA). They were subsequently processed with NBT/BCIP solution (Roche Diagnostics) according to the manufacturer's instructions to obtain the dark purple NBT-DF/BCI stain. Immunohistochemistry followingNBT/BCIP staining was performed as follows: anti-green fluorescent protein (GFP) antibody (1:500; Torrey Pines Biolabs, Houston, TX, USA) and anti--catenin (1:500; Sigma-Aldrich, St. Louis, MO, USA) were incubated with embryos overnight at 4°C, followed by incubation with an anti-rabbit or anti-mouse Alexa-conjugated secondary (1:200; Molecular Probes™; Invitrogen, Carlsbad, CA, USA). Stained embryos were embedded in 4% NuSieve® GTG low melting agarose (Fisher Scientific, Pittsburgh, PA, USA) and cut into 100-or 200-µm sections with a Vibratome® 1000 Sectioning System from Technical Products International (St. Louis, MO, USA) for imaging.