ARTICLE |
Correspondence to: Valérie Mitchell, Laboratoire de Neuroendocrinologie et Physiopathologie Neuronale, INSERM U422, 1 place de Verdun, 59045 Lille cédex, France. E-mail: mitchell@biserte.lille.inserm.fr
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Summary |
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In a attempt to improve the sensitivity of the simultaneous use of immunohistochemistry (IHC) with estrogen receptor (ER) and in situ hybridization (ISH) with a neuropeptide receptor, we first applied an existing microwave (MW) irradiation protocol for immunohistochemical detection of the estrogen receptor in frozen brain sections. Regions of interest were the preoptic area and the arcuate nucleus of the hypothalamus. ER signal was effective only after MW heating of sections in the two regions. Control sections without pretreatment exhibited no staining for ER. Second, the MW protocol was applied in a novel procedure that consists of evaluation of the expression of the galanin receptor mRNA with a radioactive riboprobe after MW pretreatment. The galanin receptor mRNA signal intensity obtained after heating was quantitatively at least as good or significantly increased according to the region, with no discernible loss of tissue morphology. Finally, we describe a novel application of MW pretreatment on the same frozen section processed with ER antibody and a radioactive galanin receptor riboprobe. The stainings for estrogen and galanin receptors were intense in many cells of the preoptic area, with very low background. These results show that both IHC and ISH can be significantly improved by subjecting frozen sections to MW heating before the double labeling. This approach may provide a potential method to answer the important question of whether or not estrogen has a direct action on the expression of a peptide receptor. (J Histochem Cytochem 49:901910, 2001)
Key Words: radioactive in situ hybridization, microwave heating, riboprobes, double labeling for receptors, brain, cryostat sections
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Introduction |
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DOUBLE-LABELING IMMUNOHISTOCHEMICAL (IHC) and in situ hybridization (ISH) technique has been used in neuroendocrinological studies during past years and has gained importance in research disciplines and, to a lesser extent, in histopathology to determine the co-localization of specific proteins and RNA sequences in tissue sections. However, the routine use of these techniques is often not possible because of technical difficulties. The microwave (MW) oven has been used for various applications: tissue fixation (
The aim of this work was to determine if the use of MW heating pretreatment was (a) efficient to enhance the sensitivity of detection of neuropeptide receptor mRNAs and (b) favorable for double detection, i.e., IHC for detection of estrogen receptor and ISH for neuropeptide receptor. For this investigation, we first experimented on the effects of MW treatment for immunohistochemical detection of ER. In a parallel experiment, we applied the same pretreatment on the detection of a neuropeptide receptor signal to verify that the signal intensity was as good or even enhanced as that obtained with routine ISH. The target RNA was the rat hypothalamic galanin receptor Gal-R1 mRNA expression, chosen as a model because Gal-R1 is expressed in many regions of the rat hypothalamus (
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Materials and Methods |
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Animals and Tissue Preparation
Four adult ovariectomized (OVX) female Wistar rats (200250 g; CERJ, Le Genest St Isle France) were used. They were maintained on a 14-hr light and 10-hr dark cycle (lights on at 0500 hr) with food and water available ad libitum. After 12 days, animals were anesthetized with 20 mg/kg ketamine and 0.2 ml/kg xylazine, then perfused intracardially with 510 ml of saline followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed and immersed in the same fixative for 2 hr. They were then washed overnight in 0.05 M Coons' veronal buffer (pH 7.4) containing 20% sucrose, embedded in Tissue-Tek (Miles Laboratories; Naperville, CA), and frozen in liquid nitrogen. Frozen 14-µm coronal sections were cut from the level of -1.8 to -4.8 mm relative to Bregma, according to the atlas of
MW Treatment
Before the IHC, ISH, or double labeling IHCISH procedures, some sections were submitted to MW treatment. MW heating was performed using a domestic Samsung MW oven. Sections were immersed in a DEPC-treated sodium citrate buffer, 10 mM at pH 6.0, for 5 min at 800 W, immediately followed by two 4-min periods at 400 W. Sections were allowed to cool in the same buffer for 45 min at room temperature. The IHC and ISH procedures were performed as usual.
IHC labeling for ER
Some sections from each animal were rinsed in 0.1 M Tris-buffered saline (TBS) and incubated for 48 hr at 4C with a mouse monoclonal ER antibody (clone 1D5; Dako, Trappes, France) diluted 1:100 with TBS containing 0.3% Triton. A group of sections was heated by MW as described above and the other group of adjacent sections was not pretreated. Antigenantibody binding sites were visualized using biotinylated goat anti-mouse serum (1:200, for 90 min; Caltag Laboratories, Burlingame, CA), followed by a peroxidase-conjugated avidin (Vector Laboratories; Burlingame, CA). Sections were processed for the DAB reaction as usual.
The specificity of the ER antibody has been previously checked in human tissues ( (
35S-Labeled Gal-R1 Receptor Complementary RNA Probe
The plasmid vector pBluescript IIKS containing a HindIII/BamHI fragment of 1600 bp of the full-length rat Gal-R1 receptor (
In Situ Hybridization
ISH was performed following our well-characterized routine procedure (
The controls for specificity of the probe have been previously checked (
Quantitative Analysis of ISH Labeling
All sections were processed together during hybridization and autoradiographic procedures. Quantitation of Gal-R1 signal intensity was performed by light microscopy under darkfield illumination to evaluate the pattern and intensity of the hybridization signal. The estimation of the Gal-R1-labeled cells was performed by using the azure blue nuclear stain. Analysis was conducted on the entire preoptic area and arcuate nucleus according to the Swanson rat brain atlas (plates 1630). Four tissue sections per area of interest from each animal were analyzed (about 5000 cells for the preoptic area and 2000 for the arcuate nucleus). The quantification of the Gal-R1 grain density was performed using an image-analysis program (Densirag; Biocom, Les Ulis, France) interfaced to an Axiophot microscope (x60 epiillumination darkfield objective ; Carl Zeiss, Göttingen, Germany) run on a PC computer with a high-resolution camera.
The mean density of grains per cell (±SEM) was calculated. Differences among levels of Gal-R1 receptor mRNA expression in the pretreated and control groups were assessed with a Student's t-test and were regarded as significant when p0.05.
Double Labeling for ER and Gal-R1
A group of sections was submitted to the MW treatment and was then processed for the double labeling ER and Gal-R1. After the IHC labeling for ER and the DAB reaction as described above, sections were rinsed in TBS and then processed for ISH labeling as also described for detection of Gal-R1 mRNA expression. After a 30-day exposure time, sections were revealed, fixed, and observed under the Axiophot microscope. The Gal-R1 signal intensity was quantified (see above) and the results were expressed as mean density of grains per cell ± SEM.
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Results |
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Preservation of Tissue Morphology with MW Treatment
The Nissl staining showed characteristically intense dark blue nuclear staining of chromatin on a colorless background (Fig 1) in many cell bodies. MW pretreatment of sections that had undergone ISH for Gal-R1 had negligible effects on cell morphology (Fig 1A and Fig 1B) in comparison with control sections without any pretreatment (Fig 1C and Fig 1D). It demonstrated an acceptable architectural preservation of pretreated sections.
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Effect of MW Treatment on IHC ER Staining
Under OVX conditions, MW irradiation before the IHC produced staining with the clone 1D5. The signal was intense in many cells of the preoptic area (Fig 2A) and in some nuclei of the arcuate nucleus (Fig 2C). Background was negligible. On sections not pretreated with MW, labeling for ER was undetectable in the two regions of interest (Fig 2B and Fig 2D).
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Specificity of Gal-R1 ISH on MW-treated tissue sections
The localization of positive signal from MW-heated and control sections obtained with antisense Gal-R1 mRNAs was consistent with that previously published (
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The sense probe, which was used at equivalent specific activity in the same concentration as the antisense probe, gave no hybridization signal. RNase pretreatment completely abrogated hybridization signals obtained with the antisense probe.
Effect of MW Pretreatment on Hybridization Efficiency
The hybridization signal obtained from pretreated tissue sections with MW oven heating was compared to that obtained from control sections without pretreatment after a 30-day exposure (Fig 3 and Fig 4). An important observation was that the background signal obtained after MW pretreatment was not significantly increased in comparison with that of control sections. In the preoptic area, all the slides heated by MW pretreatment showed increased hybridization signal compared with the control slides (Fig 3A, Fig 3B, and Fig 4A; 39.72 ± 1.97 vs 32.77 ± 1.23). This difference was significant (p<0.05). In contrast, in the arcuate nucleus no significant difference was noted between the control group and that preheated (Fig 3C, Fig 3D, and Fig 4; 19.92 ± 1.21 vs 22.21 ± 1.72; p<0.05).
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MW Pretreatment Improves Double-labeling Efficiency
As illustrated in Fig 5, MW-pretreated double-labeled sections for ER and Gal-R1 exhibited three populations of labeled cells: ER-immunostained nuclei, Gal-R1 mRNA-expressing cells, and double-labeled ER-Gal-R1 cells. In the preoptic area, the double labeling concerned many cells, as shown in Fig 5A, whereas ER-Gal-R1 cells were sparse in the arcuate nucleus (Fig 5B). The mean optical density for Gal-R1 mRNA as quantified in double-labeled cells of the preoptic area was 26.26 ± 0.47.
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Discussion |
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Our results first confirm that, as in other tissues, the MW procedure before the IHC is very favorable to obtain staining with the ER antibody from fixed frozen sections of hypothalamus. Second, this work demonstrates that the MW preheating before the ISH improves the sensitivity of Gal-R1 expression in the preoptic area. Finally, we show that MW treatment is helpful for double-labeling techniques that originally associate the simultaneous detection of steroid receptor by IHC and neuropeptide receptor mRNA by ISH from frozen brain sections.
Since the initial description of the MW procedure by
Our results show an analogous effect of enhancement of the ISH autoradiographic signal composed to that previously observed in the literature (
The mechanisms of enhancement of the IHC or ISH signal had previously been speculated on and discussed in the literature: better penetration of the probe to the target by unmasking the crosslinking effects of formaldehyde fixation and denaturation of protein and nucleic acid structure. In IHC, when MW heating is performed on unfixed fresh tissue, this pretreatment appears to preserve antigens in sections by increasing their resistance to extraction during subsequent aqueous fixation and IHC processing (
In conclusion, MW heating demonstrates its efficiency for hybridization labeling of galanin receptor in frozen brain tissue. This is applied to the double labeling by IHC and ISH techniques on the same section and thus may help detect low levels of mRNA together with difficult detection of nuclear antigens. This protocol appears particularly suitable for simultaneous evaluation of the expression of two receptor states. This study provides evidence of a distinct expression of estrogen receptor in Gal-R1 cell populations of the preoptic area and arcuate nucleus, and of the fact that estrogens may target its -type receptor in populations expressing receptors for neuropeptides of hypothalamic regions involved in reproduction.
Received for publication November 13, 2000; accepted February 17, 2001.
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