Improved In Situ Hybridization: Color Intensity Enhancement Procedure for the Alkaline Phosphatase/Fast Red System

In situ hybridization (ISH) studies have commonly utilized digoxigeninlabeled probe followed by incubation with anti-digoxigenin antibody conjugated with alkaline phosphatase (AP) and color development using an AP substrate. Among the substrates used in the protocol, Fast Red produces a reddish signal (3). We have found that one of the major drawbacks of using Fast Red is that color intensity is generally weak, and a prolonged incubation time and/or a secondary antibody is required for signal amplification. Moreover, when secondary antibody is used, the background is increased as well as the specific signal. By using oligonucleotide probes against the mRNA of the nicotinic acetylcholine receptor (AChR) α-subunit in cultured chick embryonic muscle cells, we found that color intensity can be dramatically increased by adding sodium chloride in the color development solution without secondary antibody amplification, and that the degree of signal augmentation is correlated with sodium chloride concentration. During myogenesis, mononucleated myoblasts fuse to form multinucleated myotubes. Nuclei of both myoblasts and myotubes express the mRNAs of AChR subunits (α, β, γ and δ) in a differential fashion (1,4). These phenomena can be easily studied in culture. Chick muscle cultures were prepared by obtaining myoblasts from the limbs of 11-day-old chick embryos. The cells were then grown on collagen-coated plastic coverslips (3 cm2) in 35-mm culture dishes containing 2 mL of MEM supplemented with Earle’s salts, L-glutamine, 10% horse serum and 5% chicken embryo extract (CEE) for the first two days, followed by a change of medium containing 2% CEE for the next several days. The fibroblast population was reduced by pre-incubating the cells in petri dishes followed by plating these into 35-mm collagenized dishes containing coverslips. The majority of fibroblasts attached to the collagen-free surface and were thereby removed, while only a small number of them remained in the culture. Since fibroblasts do not express AChR, and are morphologically distinguishable from muscle cells, they may serve as controls for nonspecific binding of the probe. After three days in culture, cells were rinsed with 1× phosphatebuffered saline (PBS)/0.1% bovine serum albumin (BSA), followed by fixation with 4% paraformaldehyde for 15 min and two washes with 1× PBS (5 min each). Cells can be kept in 70% ethanol until hybridization. Oligonucleotide probes were labeled at the 3′-end with digoxigenin using terminal deoxyribonucleotide transferase (TdT; Promega, Madison, WI, USA). The α-specific probe mixture contains α exon probe (α E) and α exon/intron probe (α EI). The probe α E (5′-GTATTCCCGGAACAGGTCATCCACCAGGCGCGTCTCGTG-3′) is located in the exon 2 region, while the probe α EI (5′CAGGGCCGGCCCGGCTGTGAGTGACAAAATGGCATCAGTGAGAGGGGT-3′) is located in intron 1 and exon 2 region. Another oligonucleotide probe, denoted as Ep (5′-GAACGAGACTTGGCAGATAAGGATGAGTCC-3′), is specific to a mouse neuronal glutamate receptor subunit (NMDAε2) and was used as a negative control. The labeling reaction was carried out at 37°C for 2 h in a TdT buffer solution containing 0.15 mM dATP, 50 μM digoxigenin11-dUTP, 140 pmol oligonucleotides and 30 U TdT. The reaction was terminated by adding 10 mM EDTA, 1 μg/μL glycogen, 0.3 M LiCl and 2.5 vol of absolute ethanol for precipitation. After washing with cold 70% ethanol (4°C), the pellet was resuspended in 100 μL TE (10 mM Tris-HCl [pH 7.6]/1 mM EDTA) buffer containing 0.1% sodium dodecyl sulfate (SDS) and then heated to 70°C followed by vortex mixing. Probes were evaluated by the dot blotting method as follows: 1 μL TE buffer was added to 1 μL of each newly synthesized probe and mixed. One microliter of the mixture at different dilutions was spotted onto a nylon membrane (Boehringer Mannheim, Mannheim, Germany). The membrane was air-dried for 15 min, soaked in Buffer A (0.1 M Tris-HCl, pH 7.5/0.15 M NaCl) for 5 min, followed by 2% blocking reagent (Catalog No. 1093657; Boehringer Mannheim) for 1 h and then incubated for another hour with anti-digoxigenin-AP Fab fragment (Catalog No. 1093274; Boehringer Mannheim) at a 1:1000 dilution containing 2% blocking reagent. The membrane was washed with Buffer A for 20 min with 5 changes and rinsed for 2 min with Buffer B (0.1 M TrisHCl, pH 9.5/0.1 M NaCl/50 mM MgCl2). Dot blots were developed by using Buffer B containing 0.5 mM nitro blue tetrazolium (NBT) and 0.1 mM xphosphate/5-bromo-4-chloro-3-indolylphosphate (Boehringer Mannheim). When using two different probes, color intensity was compared to ensure that probes are labeled with a similar degree of intensity. Probes were stored at -20°C. Immediately before use, the probes were heat-denatured at 95°C for 5 min followed by chilling with ice. Before ISH, cells on the coverslip were treated for 10 min each using the following solutions: 1× PBS/5 mM MgCl2 at room temperature (RT); 0.2 M Tris-HCl, pH 7.7/0.1 M glycine at RT; 0.1 M ethanolamine/0.01 M acetic anhydride at RT; and 2× standard saline citrate (SSC)/50% de-ionized formamide at 65°C. ISH was performed at 37°C overnight in a Saran Wrap®-sealed moisture chamber. Pre-hybridization was not required, as shown previously for radiolabeled probes (2). The hybridization solution contained 5% dextran sulfate, 50% formamide, 0.08 M Tris-HCl (pH 7.5), 0.2% SDS, 0.1% sodium pyrophosphate, salmon sperm DNA (0.3 μg/μL), E. coli tRNA (0.3 μg/μL), 0.1 M dithiothreitol (DTT), heparin sulfate (0.2 μg/μL), NaCl (0.6 M) and 4 mM EDTA. Fifty microliters of hybridization solution containing 1.5 pmol digoxigenin-labeled antisense oligonucleotide probe(s) were used in each sample. After hybridization, the coverslips were quickly rinsed with 1× SSC to remove the carryover, followed by four washes, each of 20-min duration, with 1× SSC/50% formamide/20 mM DTT at 37°C and one wash at RT with 1× SSC/20 mM DTT for 30 min.