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Correspondence to: A.R. Lisowski, Global Toxicology, Pharmacia, 4901 Searle Parkway, Skokie, IL 60077. E-mail: andrew.r.lisowski@monsanto.com
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Summary |
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In this study we evaluated whether storing non-deparaffinized sections can affect the detection of specific mRNAs by radioactive in situ hybridization (ISH). Using a standard ISH protocol, we hybridized serial sections of paraffin blocks stored for different periods of time with 33P-labeled riboprobes specific for rat Type III collagen and matrix metalloproteinase-2 (MMP-2). Signal intensities were evaluated using a phosphorimager and by blinded microscopic examination. For slides hybridized with the Type III collagen riboprobe, signal intensities measured with the phosphorimager or evaluated by microscopic examination were negatively correlated with the storage period of the sections. For slides hybridized with the MMP-2 riboprobe, differences in signal intensity could be detected, albeit inconsistently, with the phosphorimager, although microscopic examination consistently indicated stronger signals in freshly sectioned slides compared to slides stored for 2 weeks or more. We concluded that it was preferable to use recently prepared sections for trying to locate mRNAs in paraffin-embedded tissues by ISH. In addition, our results suggest that quantifying signal intensity using a phosphorimager is feasible for abundant mRNAs or when large differences in expression are anticipated.
(J Histochem Cytochem 49:927928, 2001)
Key Words: in situ hybridization, storage time, type III collagen, MMP-2, phosphorimaging
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Introduction |
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SUCCESSFUL DETECTION and localization of mRNAs in histologic sections by in situ hybridization (ISH) require the use of well-preserved and/or adequately fixed tissues to minimize mRNA degradation (
To obtain our histological sections, we serially sectioned paraffin blocks at different times so that the resulting serial 4-µm sections would be stored for 1, 4, 9, 14, 20, 27, or 35 days, respectively. Tissue sections were stored in a light-resistant closed box at room temperature. All tissues used were fixed in 10% neutral buffered formalin for 24 hr and routinely processed and embedded. These tissues were collected from in vivo rat studies that had received prior approval by the Institutional Animal Care and Use Committee, and consisted of experimental skin wounds collected at various time points, experimental myocardial infarcts, and fibrotic lungs from rats treated with bleomycin. To evaluate whether the nature of the mRNAs could affect the results, both Type III collagen and matrix metalloproteinase-2 (MMP-2) mRNAs were detected by radioactive ISH using an excess of 33P-labeled riboprobes. Type III collagen mRNA is abundant in the granulation tissue of skin wounds and in bleomycin-induced pulmonary fibrosis, while MMP-2 is expressed at low to moderate levels in skin wounds and myocardial infarcts (
The temporal and spatial expression of Type III collagen and MMP-2 mRNAs in skin wounds, experimental myocardial infarcts, and bleomycin-induced pulmonary fibrosis was consistent with published literature reports (
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Our results indicated that for radioactive ISH assays to detect specific mRNAs in formalin-fixed, paraffin-embedded tissues, the use of freshly sectioned slides provides better signals. Therefore, it is recommended to use freshly sectioned slides (i.e., stored for 4 days or less) for trying to locate specific mRNAs in formalin-fixed, paraffin-embedded tissues by radioactive ISH. Although this was not demonstrated in the present study, it is likely that lower signals were detected in slides stored for a significant period of time before hybridization because of slight degradation of the targeted mRNAs in paraffin sections stored at room temperature. It is not known whether storing the sections under different conditions could have prevented this decrease in mRNA detection. Assessment of signal intensity using a phosphorimager was useful to demonstrate and quantify the decrease in signal detection with storage period. However, the phosphorimager data were consistent only for the mRNA present in abundant quantity (i.e., Type III collagen mRNA), whereas differences were more inconsistent for the mRNA present in low to moderate amounts (i.e., MMP-2 mRNA). These results suggested that quantitative radioactive ISH in formalin-fixed, paraffin-embedded tissues by exposing hybridized sections to a phosphorimager would be reliable only for abundant mRNAs or when large differences in mRNA expression between sections are anticipated.
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Footnotes |
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Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 27, 2001.
Received for publication November 30, 2000; accepted February 16, 2001.
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