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This study addressed the question of whether radioactive hybridization signal intensities are reduced in combined isotopic and non-isotopic double in situ hybridization (DISH) compared with those in single in situ hybridization (ISH). Non-isotopic Digoxigenin (Dig)-labeled hybrids were detected using an alkaline phosphatase (AP) enzymatic reaction which results in NBT/BCIP-salt precipitation that could shield S35-radiation from penetrating to the surface. Sections were plastic coated of with 2% parlodion to prevent a chemical reaction between AP and developer during processing of the photosensitive emulsion, which could further reduce radioactive hybridization signal detection by autoradiography. We used DISH with a hybridization cocktail of radioactive S35- and Dig-labeled GAD67 cRNA probes. In order to avoid competition for the same complementary sequence, the probes were directed towards different sequences of the glutamic acid decarboxylase (GAD67) mRNA, resulting in co-detection of isotopic and non-isotopic hybrids in close to 100% of GAD67 positive cells. Quantitation of autoradiograms showed that there was no reduction of autoradiographic signal intensity from S35-labeled hybrids in the presence of Dig-labeled hybrids. Plastic coating of single or dual hybridized sections did not reduce the radioactive signal intensity. When mRNAs for nicotinic acetylcholine receptor (nAChR) subunits were detected with subunit specific S35-labeled cRNA probes in GAD67 hippocampal interneurons the total numbers of nAChR subunit expressing cells remained the same in single or double hybridized sections even for low abundant mRNAs. Together, these results indicate that combined radioactive and non-radioactive DISH does not interfere with the detection of the radiation signal from the S35-labeled hybrids, and neither specificity nor sensitivity is compromised.
In situ hybridization (ISH) is a powerful tool to study the anatomical locations of mRNAs. Both cRNA or cDNA probes are useful tools for the detection of complementary transcripts, and result in high target specificity and sensitivity, and allow the detection of transcripts in small nuclei and even single cells (Cox et al., 1984, Winzer-Serhan et al., 1997). In order to detect the expression of more than one transcript, which is especially valuable for the identification of specific cell populations, probes for different target mRNAs are labeled with different reporters and combined for “double” in situ hybridization (DISH). Several studies have used a combination of isotopic and non-isotopic protocols, where one probe is labeled with S35-UTP and the other with digoxigenin (Dig)-UTP, respectively (Winzer-Serhan and Leslie 1997, Bizon et al., 1999, Day et al., 1999, Stone et al., 1999, Hohmann and Herkenham, 2000, Lin et al., 2002, Azam et al., 2003, Lu et al., 2003, Loughlin et al., 2006, Watts and Sanchez-Watts 2007. O’Leary et al., 2008, Son and Winzer-Serhan 2008). The benefit of this method is twofold; it takes advantage of the sensitivity of radioactive ISH, and it provides a permanent record so that sections can be reanalyzed years later (Hohmann 2006).
In this protocol, radiolabeled hybridization signal is detected by conventional autoradiography; the hybridized sections are apposed to film to create an autoradiographic image, followed by emulsion coating for the detection of silver grains under darkfield microscopy (Normand and Bloch, 1991, Miller et al., 1993, Trembleau et al., 1993). In most protocols, the detection of the non-radioactive Dig-hybridization signal requires the use of an alkaline phosphatase (AP)-linked antibody directed against Dig followed by an enzymatic color reaction often using the di-tetrazolium salt nitroblue tetrazolium chloride (NBT) in combination with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) as substrates which results in an insoluble dark purple precipitate, and the hybrids are then visualized with light-field microscopy.
It is generally believed that the radioactive hybridization signal is more sensitive than the non-radioactive signal (Chotteau-Lelièvre et al., 2006), which is why for DISH the lower abundant mRNA is usually detected with a S35-labeled probe, and the more abundant message with Dig-labeled probe. Although several studies have pointed out that there is no loss of signal (Normand and Bloch, 1991, Kerner et al., 1998, O’Leary et al., 2008), concerns about quenching of the radioactive hybridization signal by the non-radioactive one detected with NBT/BCIP remain (Daye et al., 1999). Because the colorimetric reaction product is deposited over the entire cell body, the insoluble salt precipitate could block the radioactive S35-signal from effectively penetrating to the surface, or reduce the radioactivity getting through. However, this is essential for the reaction with the radiosensitive emulsion placed over the sections, and therefore, the formation of silver grains. Thus, if quenching occurred it could reduce the signal derived from S35-labeled probes, which could be especially problematic for detecting low abundant targets, and in the worst case could create false negative results. Therefore, some studies have gone to great lengths to avoid interference when using double-labeling protocols (Chiu et al., 1996). In addition, the sections are often coated with a plastic film prior to coating with the emulsion, to protect sections from darkening during the development of the emulsion (Miller et al., 1993). This additional plastic film could further shield the radioactive signal and add to the quenching problem associated with combined isotopic/non-isotopic ISH.
Signal reduction due to quenching could also affect other double labeling protocols where a combination of isotopic and non-isotopic markers is used, such as radioactive ISH combined with immunohistochemistry. Therefore, in this study we wanted to address the question of whether quenching of the S35-radioactive hybridization signal by a non-radioactive colorimetric detection method occurs. Furthermore, we wanted to determine if plastic-coating of the sections with parlodion decreases the autoradiographic signal intensity of the S35-labeled hybrids, and if S35-hybridization signals from low abundant transcripts are reduced or lost by the DISH procedure.
Animal procedures were approved by the Institutional Laboratory Animal Care Committee according to the rules of the Texas A&M University, and consistent with National Institute of Health guidelines. All efforts were made to reduce the number of animals used in this study. Young adult male and female Sprague-Dawley rats (Harlan Inc., Houston, TX) were kept on a 12 h light/dark cycle with free access to food and water. The animals were anesthetized with isoflurane, decapitated, and brains were immediately removed, frozen in −20°C isopentane, and stored at −80°C until use. Consecutive frontal brain 20 μm thick sections from three different animals (one male and two females) were cut on a cryostat, and thaw-mounted onto poly-L-lysine coated slides. The sections were postfixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 for 1 h at room temperature (RT), washed in PB, air dried and stored desiccated at −20°C until use.
A template for rat glutamic acid decarboxylase (GAD67, M76177) mRNA (185–650; 466bp) was generated by RT-PCR (forward primer: 5′-ATGGCATCTTCCACGCCTTCG-3′, reverse primer: 5′-CCAAATTAAAACCTTCCATGCC-3′), subcloned into pPCR-Script Amp SK (+) according to the manufacturer’s instructions (Stratagene Inc., La Jolla, CA), and sequenced to verify the sequence. Dig-labeled GAD67 antisense cRNA probes were synthesized using Dig-RNA labeling mixture (Roche Applied Science, Indianapolis, IN) with T7 RNA polymerase (Applied Biosystems/Ambion, Austin, TX). In addition, a PCR product for GAD67 (1198–1474; 277 bp) was used as a template to synthesize a S35-labeled GAD67 antisense cRNA probe by in vitro transcription after adding the T3 promoter sequence to the reverse primer (forward: 5′-TTATGTCAATGCAACCGCAGGC-3′, reverse: 5′-AATTAACCCTCAAAGGN(13)ACACATCTGGTTGCATCCTTGG-3′).
Plasmids containing cDNAs for nicotinic acetylcholine receptor (nAChR) subunits (kindly provided by Dr. J. Boulter, UCLA, CA), subcloned into pBluescript II SK between T3 and T7 promoter sites, were used as templates. Radiolabeled cRNA probes for α2 (1931 bp), α3 (1858 bp), α4 (2110 bp), α5 (1607 bp), α7 (2100 bp), β2 (2196 bp) and β4 (2522 bp) were synthesized in the presence of S35-UTP (PerkinElmer, Boston, MA). Full-length probes were further subjected to alkaline hydrolysis to yield products with an average size of 600 bp according to the method by Cox et al. (1984). Full-length cRNA probes (except for the α2 cRNA probe, which was not hydrolyzed) were incubated by adding one volume of carbonate buffer (80 mM NaHCO3 and 120 mM Na2CO3) freshly prepared in sterile DEPC-treated water just prior to use. The hydrolysis time was calculated according to the formula t = (Lo − Lf)/iLoLf, (Lo = probe length in kilobase [kb], Lf = final length in kb, i = 0.11). The reaction was stopped precisely at the calculated time by adding 1/3 vol. of 3 M sodium acetate pH 6.0 and 1/20 vol. of 10 % glacial acetic acid to the reaction mixture.
The ISH procedure follows the method previously described in detail by Winzer-Serhan et al. (1999). If not otherwise stated, the materials were purchased from Sigma. Sections were pretreated with 0.1 μg/ml proteinase K for 30 min at RT, rinsed in 0.1 M triethanolamine (TEA) (Fisher Scientific) pH 8 for 2 min and acetylated with 0.1 M TEA with 0.25% acetic anhydride (EMD Chemicals, Gibbstown, NJ) for 10 min, dehydrated through graded ethanols (50, 70, 95 and 100 %) and air-dried. Sections were then incubated for 18 h at 60°C with a 1:1 dilution of Dig-labeled antisense GAD67 cRNA probe (0.1 μg/ml) and S35-labeled antisense probes (2×107 cpm/ml), or S35-labeled probes alone (1×107 cpm/ml) in hybridization solution (50% formamide, 10% dextran sulfate, 500 μg/ml tRNA, 10 mM dithiothreitol, 0.3 M NaCl, 10 mM Tris, pH8.0, and 1 mM EDTA, pH 8.0). After hybridization, sections were incubated with RNase A (20 μg/ml) for 30 min at 37°C, followed by two 5 min washes in 2x standard saline citrate buffer (SSC), two 10 min washes with 1x and 0.5x SSC at RT, and a 30 min wash in 0.1x SSC at 65°C. Single S35-labeled sections were dehydrated and air dried at this point.
For DISH, after the hot wash, the slides were incubated in Genius buffer (GB) (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) for 5 min, followed by 30 min incubation in 5% nonfat dry milk (Carnation, Nestle Inc.) in GB plus 0.25 % Triton-X at RT. The AP-conjugated anti-Dig Fab antibody (sheep) (Roche Applied Science, Indianapolis, IN), prepared as 1:1,000 dilution in GB, was applied to the sections and slides were incubated for 3 h at RT. The slides were washed three times for 1, 5, and 10 min in GB. Slides were incubated with color reagent [200 μl of NBT/BCIP stock solution (18.75 mg/ml NBT, 9.4 mg/ml BCIP) in 10 ml of 100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.5] at RT overnight. The slides were washed twice in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 and once in double de-ionized water, dehydrated with brief dips in graded ethanols (50, 70, 95, and 100%) and air-dried. The sections were apposed to Kodak Biomax MR film for an appropriate period of time. After film development, DISH processed slides were coated with 2% parlodion (SPI-Chem, West Chester, PA) in isoamylacetate and dried at RT over night. Single and dual hybridized sections were dipped in liquid NTB emulsion (VWR, West Chester, PA). After an appropriate exposure period, slides were developed in a 1:1 solution of Kodak D19 developer and deionized water for 4 min at 16°C, briefly washed in deionized water (16°C) and fixed with Kodak Professional fixer for 10 min at 16°C, and rinsed in running water for 20 min. Single S35-labeled sections were counterstained with Cresyl Violet, dehydrated in graded ethanols, air dried and cover-slipped, DISH processed slides were air-dried and cover-slipped with DPX. Note: parlodion-coated slides were not dehydrated in histoclear because it removes the parlodion coating.
Four coronal frontal brain sections, 320 μm apart from each other, from three animals were used for analysis. Autoradiographic images and relative optical density (ROD) were obtained using a computer-based image analysis system (MCID, Imaging Research Inc. St. Catherine, Canada; now InterFocus Imaging Ltd, UK). A calibration curve of radioactivity (nCi/g wet weight tissue) versus optical density was generated using [14C] standards of known radioactivity (30–862 nCi/g tissue weight, GE Healthcare Bio-Science Corp. Piscataway, NJ), and regional values of optical density were converted to values of radioactivity. Data were analyzed using a Student t-test, and significance was defined as p ≤ 0.05.
To determine the sensitivity of the S35-hybridization signal in single and dual ISH, the sections were analyzed by darkfield microscopy, and the radioactive S35-hybridization signal (silver grains) was counted in positive neurons located in different fields of the hippocampus with the aid of the analyzing software DP-Manager (Leeds Instruments, Irving, TX). A positive cell was defined as ≥12 silver grains overlying a stained cell with an estimated area of 100 μm2, which was more than 4x higher than the average number of silver grains considered as background (2.5 ±1.56/100 μm2). Background signal was determined with an α7-S35-sense probe and verified by counting the number of silver grains in a part of the sections known not to express the target mRNA. For the statistical analysis, results from dorsal sections were analyzed using the Student t-test.
Photomicrographic images were obtained using an Olympus BX51 microscope (X4 and X20, NA 1.0) equipped with a DP7-1 digital camera (Leeds Instruments, Irving, TX) and adjusted for brightness and contrast using the Adobe Photoshop 9.0 image-editing software package (Adobe System, Mountain View, CA).
To compare the sensitivity of the S35-hybridization signal between single and dual ISH, adjacent brain sections were hybridized with the S35-GAD67 probe alone or in combination with the Dig-GAD67 probe. Both GAD67 probes targeted the same GAD67 mRNA but the probes were directed towards different sequences on the transcript. Thus, hybridization with the two probes should not interfere with each other, but both hybrids, Dig-GAD67 and S35-GAD67, should be located on the same mRNA strand. The hybridization patterns derived from the Dig-GAD67 and S35-GAD67 probes were identical in all brain regions studied as determined by light-field (Fig. 1A, B, C) and darkfield (Fig. 1A′, B′, C′) microscopy, respectively. Silver grains, indicating hybridization with the S35-GAD probe, were overlaying cells marked by the purple colorimetric reaction product derived from the Dig-GAD67 hybrids in the hippocampus, cingulated cortex, and reticular nucleus of the thalamus (Fig. 1a, b, c). Quantitative analysis of co-expression was done in the hippocampus because the scattered distribution of GABAergic interneurons in this structure facilitates the analysis. There was close to 100% colocalization of hybrids derived with the two different probes in hippocampal CA1 (98.37% ± 0.83%), CA3 (98.45% ± 1.0) and dentate gyrus (DG) (99.21% ± 1.14%) in Dig-GAD-positive neurons.
We next determined if the colorimetric reaction product from Dig-GAD67 hybrids or the parlodion coating quenched the radiation emitted by the S35-GAD67 hybrids, which would result in reduced radioactivity reaching the film. The autoradiographic images showed no obvious difference in S35-hybridization signals between adjacent sections processed for single or dual ISH, before or after parlodion coating (Fig. 2A). Quantitative analysis of three different regions representing areas of low, moderate and high expression intensities indicated that the S35-GAD67 signal was equal in single and dual ISH in cingulate cortex (231.0 ± 28.04, 238.7 ± 27.94 nCi/g, respectively), caudate putamen (849.7 ± 68.62, 843.3 ± 92.72 nCi/g, respectively) and in the reticular thalamic nucleus (1199.7 ± 103.82, 1150.7 ± 135.30 nCi/g, respectively). After coating the sections with 2% parlodion, the radioactive signal intensity remained unchanged for single and dual ISH in cingulate cortex (241.7 ± 24.52, 221.0 ± 14.51 nCi/g, respectively), caudate putamen (837.3 ± 78.16, 817.3 ± 80.12 nCi/g, respectively), and the reticular thalamic nucleus (1147.0 ± 116.43, 1109.0 ± 79.15 nCi/g, respectively) resulting in no statistically significant difference (Fig. 2B).
There was no evidence of quenching of the S35-GAD67 hybridization signal by the Dig-GAD67 reaction product. However, GAD67, the synthesizing enzyme of Gamma-aminobutyric acid (GABA), is abundantly expressed in GABAergic neurons. Often DISH is used to detect mRNA transcripts in specific cell populations, and in general, the less abundant mRNA is detected with an isotopic probe. Therefore, we wanted to address the question if the colorimetric reaction product from Dig-hybridization with a probe targeting a high abundant transcript such as GAD67 would interfere with detecting low abundant mRNAs with S35-labeled probes. Neuronal nAChRs are pentamers composed of alpha and beta subunits (Sargent, 1993). A number of different nAChR subunits are detected in hippocampal GABAeric interneurons, with some such as α7 and β2 exhibiting high and others such as α3, α4 and β4 low expression intensities (Son and Winzer-Serhan, 2008). To determine if the Dig-color precipitate quenches radiation from S35-hybrids we compared the number of S35-positive nAChR subunit expressing neurons after dual and single ISH. If quenching occurred, then the number of S35-positive neurons should be lower, especially for low abundant transcripts.
Typically, the expression of α4 mRNA is weak in adult hippocampus, especially when compared to the robust α4 expression in cortex and thalamus. However, numerous scattered cells exhibiting low α4 mRNA expression were found in all CA1 and CA3 strata and in the DG in single (Fig. A, a) and dual ISH (Fig. 3B, b). Most, but not all, of the α4 expressing scattered cells co-expressed GAD67 mRNA transcripts in the hippocampus (Fig. 3c). When the number of α4-expressing cells were counted in single and double labeled sections there was no significant difference (8.1 ± 0.79 mm−2, 7.8 ± 0.44 mm−2, respectively, p=0.57).
We also analyzed the number of hippocampal cells expressing mRNAs for other nAChR subunits in single and dual ISH in the hippocampus. There was no significant difference detected for any of the subunits (Fig. 4).
It had been suggested that dual ISH using a combination of isotopic and non-isotopic labeled probes could result in reduced detection of the radioactive hybridization signal, due to quenching by the non-radioactive detection product (Ozden et al., 1990). Although no published study has systematically addressed this question, concerns about the validity of data generate with non-radioactive/radioactive DISH remain to this date, particularly those describing lack of co-expression (negative data). Therefore, we wanted to determine if quenching occurs in a combined isotopic/non-isotopic standard DISH protocol when using cRNA probes.
In order to evaluate if the non-radioactive ISH signal interferes with the radioactive signal, we generated two probes, both directed against GAD67 mRNA but towards different sequences of the transcripts. This allowed us to compare the sensitivity and specificity of the probes, and to quantify the radioactive hybridization signal from autoradiograms derived from single and dual hybridized sections. We also determined whether S35-hybridization signals from low abundant transcripts, such as those for nAChR subunits in the hippocampus, would be reduced by DISH. The major finding is that DISH, as described in the present study, did not result in a significant reduction of either strong or weak S35-hybridization signals for either high abundant transcripts such as GAD67 mRNA or low abundant transcripts such as nAChR subunit mRNAs, and no reduction in signal intensity due to Parlodion coating was detected.
In general, the patterns for both GAD67 probes were identical to each other, and are in agreement with the known distribution of GAD67 mRNA in GABAergic neurons (Ferraguti et al., 1990, Esclapez et al., 1993, 1994). The results clearly showed reliable labeling by both probes, resulting in almost 100% co-hybridization in GABAergic cells. Thus, GAD67 transcripts were consistently detected with both methods, and exhibited very little non-specific background. Furthermore, there was no difference in the S35-GAD67 hybridization signal intensity in any brain area regardless of the intensity of the Dig-GAD67 hybridization signal. Areas exhibiting high expression, and hence robust NBT/BCIP precipitation, exhibited strong S35-hybridization signal, and areas of lower GAD67 expression, and therefore weaker Dig-signal, exhibited lower S35-signal. The S35-hybridization signal intensity was not reduced by DISH, and was statistically identical between single and dual ISH. Thus, there was no evidence of quenching of the radiation coming from the S35-hybrids by NBT/BCIP precipitation.
GAD67 mRNA is abundantly expressed in GABAergic neurons; therefore, we wanted to determine if the strong NBT/BCIP-salt precipitation derived from Dig-GAD67 hybridization could interfere with low abundant transcripts. In previous studies, we and others have reported that nAChR subunits are co-expressed in GABAergic neurons in the hippocampus (Son et al., 2008, Sudweeks and Yakel, 2000). Some nAChR subunits exhibit robust expression in most GABAergic neurons (α7 and β2), others exhibit strong co-expression but in a limited number of GABAergic neurons (α2 and α5), and some exhibit low expression in GABAergic neurons (α3, α4 and β4). In the last group in particular, DISH could result in an underrepresentation of co-expression due to false negative results. In order to increase the sensitivity of the S35-signal, full-length cRNA probes were used for the detection of nAChR subunits (probe length between 1607 and 2522 bp). The number of subunit positive cells in non-principal layers of the hippocampus, where hippocampal GABAergic interneurons are located (Houser and Esclapez, 1994), was determined and compared between single and dual ISH. Although the Dig-GAD hybridization signal was robust, there was no difference in the number of nAChR subunit positive cells between the methods for any of the subunits tested. The results clearly demonstrated that even for very low abundant mRNAs such as α3, α4 and β4, the number of positively identified neurons did not change, indicating that DISH did not quench low intensity S35-signals. These results support earlier findings by O’Leary et al., (2008) who addressed the problem of quenching by grain counting analysis and demonstrated that in cells, clearly positive for nAChR subunit mRNA expression, there was no significant difference in the number of silver grains over tyrosine hydroxylase (TH) Dig-labeled compared with non-TH-labeled cells, concluding that the Dig-hybridization signal did not reduce the number of silver grains over TH-Dig-positive neurons.
However, to maximize detection sensitivity for low abundant transcripts, such as those for nAChR subunits, and to increase the signal-to-noise ratio, long (>1500 bp) S35-labeled cRNA probes were used in this and other studies (Winzer-Serhan and Leslie, 1997, Azam et al., 2003, Loughlin et al., 2006, O’Leary et al., 2008, Son and Winzer-Serhan 2008. Although, there was no evidence for S35signal reduction by DISH, it is possible that weak radioactive-signals derived from hybridization to very low abundant transcripts and/or detected with less sensitive probes, such as short cRNA or oligonucleotide probes, especially in combination with very strong Dig-signal, could be reduced to a level that is statistically indistinguishable from non-specific background levels. Thus, negative results must be interpreted with caution. However, if doubts about results indicating lack of co-expression remain, the radioactive hybridization signal can be increased by using 35S-UTP and 35S-CTP for the probe synthesis or by increasing the length of the cRNA probe in order to boost the radioactive hybridization signal.
The data provided in this study from both qualitative and quantitative analysis clearly showed that the NBT/BCIP Dig-hybridization signal did not interfere with the radioactive signal in DISH, which is in agreement with reports by others (Normand and Bloch, 1991, Kerner et al., 1998, O’Leary et al., 2008). This finding is probably the result of using highly sensitive full-length cRNA probes and a fully optimized ISH protocol (Winzer-Serhan at al., 1999). The reasons for a reduction in radioactive hybridization signal reported before are not clear (Ozden et al., 1990). It is possible that DISH with oligomeric probes which have far lower radiation intensity could result in a reduction of radioactive signal, or could be the result of degradation of target mRNAs. The results presented in this study can also have implications for other double labeling protocols where combinations of radioactive and non-radioactive detections are used.
This study was supported by NIH Grant # DA016487.
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