Journal of Histochemistry and Cytochemistry, Vol. 49, 927-928, July 2001, Copyright © 2001, The Histochemical Society, Inc.


BRIEF REPORT

Effect of the Storage Period of Paraffin Sections on the Detection of mRNAs by In Situ Hybridization

A.R. Lisowskia, M.L. Englisha, A.C. Opsahla, R.T. Buncha, and E.A.G. Blommea
a Pharmacia Corporation, Global Toxicology, Skokie, Illinois

Correspondence to: A.R. Lisowski, Global Toxicology, Pharmacia, 4901 Searle Parkway, Skokie, IL 60077. E-mail: andrew.r.lisowski@monsanto.com


  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 49:927–928, 2001)

Key Words: in situ hybridization, storage time, type III collagen, MMP-2, phosphorimaging


  Introduction
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Summary
Introduction
Literature Cited

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 (Waller and Savage 1994 ). Whereas RNA degradation in fresh tissues is a well-recognized and well-understood phenomenon, little is known about the stability of RNA in non-deparaffinized histological sections that are stored before use (Waller and Savage 1994 ). In this study, our objective was to evaluate whether storing non-deparaffinized histological sections before hybridization can affect the detection of specific mRNAs by radioactive ISH.

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 (Westergren-Thorsson et al. 1993 ; Blomme et al. 1999 ; Peterson et al. 2000 ). The protocol used for radioactive ISH was as previously described (Lu et al. 1994). Plasmids containing RT-PCR cloned cDNA fragments of rat Type III collagen and rat MMP-2 were prepared, and specific sense and antisense 33P-labeled riboprobes for both mRNAs were generated before each experiment by in vitro transcription. All serial sections of the same tissue were hybridized with the same probe in triplicate during the same assay to avoid any interassay variability. The quality of the signals was evaluated qualitatively by light microscopy after exposure to photographic emulsion for 2 or 3 weeks. The intensity of the signal was also evaluated quantitatively by exposing the slide to a phosphorimager screen and measuring the intensity of the signal.

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 (Westergren-Thorsson et al. 1993 ; Blomme et al. 1999 ; Peterson et al. 2000 ). The storage time of the paraffin sections affected the quality of the signals when tissues were hybridized with the antisense Type III collagen riboprobe. Using the phosphorimager data, the volumes of radioactive signal detected in the areas of interest were 30–50% lower in slides sectioned 35 days before hybridization compared to slides sectioned the day before hybridization (Fig 1). These results were confirmed by blind qualitative evaluation of the slides by light microscopy. The decrease in signal intensity was dependent on the length of the storage period before hybridization but was not dependent on the nature of the tissue, because results were similar in different skin wounds and in fibrotic lungs. When tissues were hybridized with the antisense MMP-2 riboprobe, differences in the signal intensity could be detected among the serial sections with the phosphorimager, but these differences were not as consistent as those seen with the Type III collagen riboprobe. However, a blinded qualitative evaluation of the slides consistently indicated that the signal was greater in freshly sectioned slides compared to slides sectioned 2 weeks or more before hybridization.



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Figure 1. Effect of storage on Type III collagen mRNA hybridization signal intensity.

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.


  Footnotes

Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 2–7, 2001.

Received for publication November 30, 2000; accepted February 16, 2001.
  Literature Cited
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Summary
Introduction
Literature Cited

Blomme EA, Zhou H, Kartsogiannis V, Capen CC, Rosol TJ (1999) Spatial and temporal expression of parathyroid hormone-related protein during wound healing. J Invest Dermatol 112:788-795[Abstract/Free Full Text]

Lu LH, Gillett NA (1994) An optimized protocol for in situ hybridization using PCR-generated 33P-labeled riboprobes. Cell Vis 1:169-176

Peterson JT, Li H, Dillon L, Bryant JW (2000) Evolution of matrix metalloprotease and tissue inhibitor expression during heart failure progression in the infarcted rat. Cardiovasc Res 46:307-315[Medline]

Waller HA, Savage AK (1994) Analysis of gene transcription in situ: methodological considerations and applications in pathology. J Histotechnol 17:203-218

Westergren–Thorsson G, Hernnas J, Sarnstrand B, Oldberg A, Heinegard D, Malmstrom A (1993) Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats. J Clin Invest 92:632-637[Medline]