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

• Published in: The journal of histochemistry and cytochemistry Vol. 49; no. 7; pp. 927 - 928
• Main Authors: Lisowski, A.R, English, M.L, Opsahl, A.C, Bunch, R.T, Blomme, E.A.G
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA Histochemical Soc 01.07.2001; SAGE Publications
• Subjects: Animals; Bleomycin; Collagen - analysis; Collagen - genetics; Collagen - metabolism; In Situ Hybridization - methods; Lung - metabolism; Matrix Metalloproteinase 2 - analysis; Matrix Metalloproteinase 2 - genetics; Matrix Metalloproteinase 2 - metabolism; Myocardial Infarction - metabolism; Paraffin Embedding; Phosphorus Radioisotopes; Pulmonary Fibrosis - chemically induced; Pulmonary Fibrosis - metabolism; Rats; RNA, Messenger - analysis; RNA, Messenger - metabolism; Skin - injuries; Skin - metabolism; Time Factors; phosphorimaging; type III collagen; storage time; MMP-2; in situ hybridization
• ISSN: 0022-1554; 1551-5044
• DOI: 10.1177/002215540104900716

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.