TECHNICAL NOTE |
Correspondence to: Kevin R. Oliver, Merck, Sharp and Dohme Res. Labs., Neurosci. Res. Centre, Terlings Park, Eastwick Rd., Harlow, Essex CM20 2QR, UK.
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Summary |
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Paraffin embedding of tissue is generally perceived to dramatically reduce RNA detectability. As a consequence, in situ hybridization on paraffin-embedded tissue is largely confined to detection of high-copy RNA species (e.g., viral RNA) and/or to detection using typically more sensitive cDNA probes or riboprobes. In this study, several procedures for in situ hybridization on paraffin-embedded rat tissue using oligonucleotide probes complementary to cellular transcripts were developed and quantitatively compared. Certain pretreatments showed marked increases in sensitivity compared to untreated sections. Furthermore, through quantitative assessment using image analysis, sensitivity of optimal pretreatments was equal to that of routinely used fresh-frozen, postfixed tissue sections. The development of such techniques permitting in situ hybridization to be carried out on paraffin-embedded tissue allows a comparison of protein and mRNA distribution to be made in adjacent sections and provides the potential for double labeling by in situ hybridization and immunohistochemistry which may not be possible on post-fixed frozen sections. (J Histochem Cytochem 45:1707-1713, 1997)
Key Words: in situ hybridization, oligonucleotide, image analysis, pretreatment, proenkephalin, rat brain
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Introduction |
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Synthetic oligonucleotides are widely used in modern molecular biology and associated disciplines. One of their many uses is in in situ hybridization, which is increasingly being used to determine the localization of specific DNA and RNA sequences in tissue sections and cells. Oligonucleotide probes have an advantage over DNA probes and riboprobes in that they are easily and readily synthesized to known sequences, thereby avoiding homology with related sequences and consequently reducing background signal. Furthermore, they are small in size and can penetrate tissue easily. However, their primary disadvantage is that their small size and scope of labeling are limited, and sensitivity is therefore greatly reduced. In most instances in which the detection of cellular messenger RNA (mRNA) species is required, mRNA degradation must be minimized, which historically necessitated the use of rapidly removed, fresh-frozen, cryostat-cut tissue, which is postfixed and stored either as frozen (dry) sections or in 95% ethanol (
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Materials and Methods |
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Preparation of Tissue
Male Sprague-Dawley rats of 230 g were terminally anesthetized with sodium pentobarbital and transcardially perfused with 500 ml 0.9% saline containing 25 U/ml heparin. Rats were subsequently perfused with the same volume of 10% formal saline. Brains were removed and stored in fixative for 72 hr before embedding in paraffin. Sagittal sections were then cut at 6 µm using a rotary microtome (Anglia Scientific; Cambridge, UK) and mounted on Fisher Superfrost/plus slides (BDH; Poole, UK). Sections were stored in dust-free conditions at room temperature (RT).
Slide Pretreatments
All slides were deparaffinized in xylene (15 min), hydrated in graded ethanols [prepared with diethyl pyrocarbonate (DEPC)-treated water], and washed twice in DEPC-treated water. From this stage, the treatments differed. For autoclaving, sections were placed in 10 mM citrate buffer, pH 6.0, and autoclaved in a standard 130C/2 bar/40 min cycle (Astell Scientific; Sidcup, UK). Sections were allowed to cool to 70C, washed for 15 min in DEPC-treated water, and dehydrated through graded ethanols. For microwaving, sections were also immersed in 10 mM citrate buffer, pH 6.0, in a clean polyethylene chamber, covered with a lid, and microwaved at full power in an 800-W microwave [Panasonic (Matsushita Electric); Uxbridge, UK] for three 5-min periods. After each 5-min period the solution was topped up to compensate for any evaporation. Likewise, sections were washed for 15 min in DEPC-treated water and dehydrated in graded ethanols. For simple heating, slides were placed in DEPC-treated water for 120 min at 90C, before dehydration in graded ethanols. For pretreatment with proteinase K, sections were treated sequentially with 0.2 M HCl (10 min), 1% Triton X-100 (90 sec), 8 µg/ml proteinase K (Boehringer; Mannheim, Germany) in 20 mM Tris, 2 mM CaCl2, pH 7.0 [for 20 min, 40 min, or 90 min, at 37C, based on optimal conditions as previously described (
For comparison, sections from fresh-frozen rat brains were probed for proenkephalin mRNA using a typical, well-characterized in situ hybridization protocol (
Design and Synthesis of Oligonucleotide Probes
An oligonucleotide probe complementary to nucleotides 388-432 of rat proenkephalin (
In Situ Hybridization
The oligonucleotide probe was labeled in a reaction mixture containing 0.4 pmol oligonucleotide, 1 x reaction buffer (Boehringer), 2.5 mM cobalt chloride, 25 U terminal deoxynucleotidyl transferase (TdT; Boehringer), and 19 µCi [35S]-deoxyadenosine 5'(-thiotriphosphate) (NEN; Hounslow, UK). Specific activity was 1250 Ci/mmol (12.5 mCi/ml) for 15 min at 37C. On cessation of the labeling reaction, dithiothreitol was added to a final concentration of 40 mM. The radiolabeled probe was subsequently purified from unincorporated nucleotides using Sephadex G-50 spin columns. Hybridization was carried out as described previously (
For qualitative histological assessment of the effect of the various pretreatments on tissue morphology, sections that had undergone in situ hybridization after the pretreatment procedures outlined above were stained with hematoxylin and eosin and examined under a Leitz DMRD microscope (Leica; Nussloch, Germany).
Evaluation of In Situ Hybridization Results
The sensitivity of the pretreatments was calculated by densitometric analysis of in situ hybridization signal on autoradiographs. All hybridizations of all sections were carried out using the same probe mixture and same washes at the same time to ensure optimal comparability. Furthermore, reference standards were used to allow interfilm comparison where required. Image analysis was carried out using a MCID computerized image analysis system (Imaging Research; St Catharines, Ontario, Canada).
Quantitative analysis was carried out on at least three sections from each brain. The hybridization signals of the sections were correlated to a standard curve obtained from exposing a series of radioactive 35S standards (obtained by serial dilution of an aliquot of the 35S-labeled proenkephalin probe) to the same film as the sections. The optical density measurements of the standards (and therefore the sections) were translated into absolute amounts of radioactive nucleotide and proenkephalin mRNA detected in attomol/mm2.
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Results |
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Effect of Pretreatment Regimens on Sensitization of In Situ Hybridization Signal
In situ hybridization signal for proenkephalin was observed predominantly in the caudate putamen (Figure 1), as previously described (
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Effect of Pretreatment Regimens on Tissue Morphology
Histological assessment was carried out on hematoxylin and eosin-stained sections that had undergone in situ hybridization for proenkephalin. Sections that had not undergone any pretreatment apart from basic deparaffinization and dehydration were regarded as the benchmark and showed characteristically intense hematoxylin staining primarily of chromatin and, to a lesser extent, the somal cytoplasm, with glial cells (e.g., oligodendrocytes and astrocytes) staining more intensely. Eosin stained the cytoplasm and myelin of white matter tracts, the latter being the most intense (Figure 3A). Sections pretreated with microwaving, heating at 90C or proteinase K treatment for 20 min had negligible effects on cell morphology (Figure 3B, Figure 3E, and Figure 3F). Chromatin structure was marginally impaired compared to controls in sections pretreated for 40 min in proteinase K but was still perfectly acceptable (Figure 3C). Very poor preservation of cell morphology was observed in sections pretreated by autoclaving or 90 min of proteinase K digestion (Figure 3D, and Figure 3G). Sections that had been autoclaved showed minimal eosin staining of the neuropil, although strong white matter fiber staining was preserved. All neuronal nuclei appeared to have disappeared from the section, although some glial nuclei remained, often in a damaged state. In some cases, the nucleoli appeared to be spared and occupied the appropriate position in the void in which the nucleus had been (Figure 3G). Sections that had been digested for 90 min in proteinase K also demonstrated absence of eosin staining in the neuropil, leaving strong eosin-stained white matter fibers (Figure 3D). Cytoplasmic hematoxylin staining was also affected and gave cells the appearance of homogenous blue nuclei in which the nuclear chromatin structure was poorly preserved (Figure 3G). The nucleoli in some cells were still apparent. Frozen postfixed sections had relatively poor morphology compared to all paraffin sections except those pretreated with 90 min of proteinase K or autoclaving (Figure 3H). Hematoxylin staining of the nucleus was very intense, and it was difficult to distinguish neurons from glia. Chromatin structure was indistinguishable. Eosin staining overall was relatively weak, and was marginally stronger in the white matter tracts. Individual fibers were much less clearly distinguishable than in any of the paraffin sections, even those subjected to severe pretreatments, such as autoclaving.
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Discussion |
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In this study we have compared an array of pretreatment regimens using quantitative densitometric techniques to determine the optimal conditions for sensitization of in situ hybridization signal on paraffin-embedded brain sections. Many methods have been documented for retrieving antigenicity in archival paraffin sections (
It is generally believed that the greatest hybridization signal is achieved using fresh tissue, cut frozen and lightly postfixed in 4% paraformaldehyde in PBS. In this study we achieved a high degree of sensitivity using this method, although the signal intensity in paraffin sections pretreated by microwaving was equally high. Assuming that the oligonucleotide probe is small enough to penetrate into the depth of the section completely and that all ß-particles reach the overlying film, it can be concluded that microwave pretreatment of paraffin tissue allows more sensitive detection of mRNA than in fresh tissue, because in this experiment we have compared routinely used 10-µm frozen postfixed sections with 6-µm paraffin sections. The reasons for enhanced sensitivity may involve several factors. First, microwaving may allow greater penetration of the probe because of its ability to disrupt crosslinked proteins formed as a result of formaldehyde fixation (
Although the microwave procedure appears to optimally sensitize the in situ signal, these data must be discussed in the light of the morphological analysis if sections are to be examined by light microscopy in addition to preliminary autoradiographic analysis. It was clear from the hematoxylin and eosin-stained sections that optimal cell morphology was maintained in sections heated at 90C in distilled water or those pretreated with 20 min of proteinase K digestion. However, these had significantly weaker signal intensities than those microwaved. Ultimately to decide on which protocol to use would depend on the absolute need of optimal morphology combined with estimated quantity of target RNA. In most circumstances, the use of the microwaving conditions outlined in this study would be entirely satisfactory. This would have the clear advantage of also allowing the potential for double labeling by immunohistochemistry and in situ hybridization on adjacent sections or on the same section, because microwaving in these conditions has been used by ourselves and others to retrieve antigenicity (
We conclude that after suitable pretreatment, such as microwaving for 15 min in citrate buffer, RNA detectability by in situ hybridization is greatly enhanced, to a greater degree than in frozen postfixed sections. This method has many advantages, including preservation of superior morphology, enhanced potential for double labeling, and more convenient and stable storage characteristics with increased longevity.
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Acknowledgments |
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We are grateful to Ms A. Jennings for technical assistance and to Dr M. Rigby for expertise in image analysis.
Received for publication February 11, 1997; accepted June 19, 1997.
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