A modified immunofluorescence in situ hybridization method to detect long non‑coding RNAs and proteins in frozen spinal cord sections

Meng,Chong; Zhao,Xin; Lao,Jie

doi:10.3892/etm.2018.6046

A modified immunofluorescence in situ hybridization method to detect long non‑coding RNAs and proteins in frozen spinal cord sections

Authors:
- Chong Meng
- Xin Zhao
- Jie Lao
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Affiliations: Department of Hand Surgery, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China
Published online on: April 10, 2018 https://doi.org/10.3892/etm.2018.6046
Pages: 4623-4628
Copyright: © Meng et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 4.0].

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Abstract

Immunofluorescence in situ hybridization (immuno‑FISH) is widely used to co‑detect RNAs and proteins in order to study their spatial distribution in cells. The present study used a modified immuno‑FISH protocol for the detection of long non‑coding RNAs (lncRNAs) and proteins in frozen spinal cord sections. The spinal cords of Sprague‑Dawley rats were harvested, frozen and sectioned (10 µm), and oligonucleotide probes and antibodies were prepared. Following antigen retrieval, dehydration, prehybridization, hybridization, post‑hybridization and immunofluorescence staining, images were captured. Antigen retrieval was performed by autoclaving or proteinase K treatment, and their effects on the hybridization signal were compared. The same sections were successfully stained by immunofluorescence. Satisfactory fluorescent signals of lncRNA and protein were obtained. The results of the present study suggest that the modified protocol of immuno‑FISH for the detection of lncRNAs and proteins in frozen spinal cord sections is effective and time‑efficient, and the required reagents are readily available.

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1	Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ, Tsai MC, Hung T, Argani P, Rinn JL, et al: Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 464:1071–1076. 2010. View Article : Google Scholar : PubMed/NCBI
2	Lee JT: Epigenetic regulation by long noncoding RNAs. Science. 338:1435–1439. 2012. View Article : Google Scholar : PubMed/NCBI
3	Zhao X, Tang Z, Zhang H, Atianjoh FE, Zhao JY, Liang L, Wang W, Guan X, Kao SC, Tiwari V, et al: A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons. Nat Neurosci. 16:1024–1031. 2013. View Article : Google Scholar : PubMed/NCBI
4	Gutschner T, Hammerle M and Diederichs S: MALAT1-a paradigm for long noncoding RNA function in cancer. J Mol Med (Berl). 91:791–801. 2013. View Article : Google Scholar : PubMed/NCBI
5	Kryger R, Fan L, Wilce PA and Jaquet V: MALAT-1, a non protein-coding RNA is upregulated in the cerebellum, hippocampus and brain stem of human alcoholics. Alcohol. 46:629–634. 2012. View Article : Google Scholar : PubMed/NCBI
6	Han Y, Wu Z, Wu T, Huang Y, Cheng Z, Li X, Sun T, Xie X, Zhou Y and Du Z: Tumor-suppressive function of long noncoding RNA MALAT1 in glioma cells by downregulation of MMP2 and inactivation of ERK/MAPK signaling. Cell Death Dis. 7:e21232016. View Article : Google Scholar : PubMed/NCBI
7	Pardue ML and Gall JG: Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc Natl Acad Sci USA. 64:600–604. 1969. View Article : Google Scholar : PubMed/NCBI
8	Fuller CE and Perry A: Fluorescence in situ hybridization (FISH) in diagnostic and investigative neuropathology. Brain Pathol. 12:67–86. 2002. View Article : Google Scholar : PubMed/NCBI
9	Nehmé B, Henry M and Mouginot D: Combined fluorescent in situ hybridization and immunofluorescence: Limiting factors and a substitution strategy for slide-mounted tissue sections. J Neurosci Methods. 196:281–288. 2011. View Article : Google Scholar : PubMed/NCBI
10	de Planell-Saguer M, Rodicio MC and Mourelatos Z: Rapid in situ codetection of noncoding RNAs and proteins in cells and formalin-fixed paraffin-embedded tissue sections without protease treatment. Nat Protoc. 5:1061–1073. 2010. View Article : Google Scholar : PubMed/NCBI
11	Meng X, Yang F, Ouyang T, Liu B, Wu C and Jiang W: Specific gene expression in mouse cortical astrocytes is mediated by a 1740bp-GFAP promoter-driven combined adeno-associated virus 2/5/7/8/9. Neurosci Lett. 593:45–50. 2015. View Article : Google Scholar : PubMed/NCBI
12	Clark MB and Mattick JS: Long noncoding RNAs in cell biology. Semin Cell Dev Biol. 22:366–376. 2011. View Article : Google Scholar : PubMed/NCBI
13	Qureshi IA, Mattick JS and Mehler MF: Long non-coding RNAs in nervous system function and disease. Brain Res. 1338:20–35. 2010. View Article : Google Scholar : PubMed/NCBI
14	Namekawa SH and Lee JT: Detection of nascent RNA, single-copy DNA and protein localization by immunoFISH in mouse germ cells and preimplantation embryos. Nat Protoc. 6:270–284. 2011. View Article : Google Scholar : PubMed/NCBI
15	Sui QQ, Zhu J, Li X, Knight GE, He C, Burnstock G, Yuan H and Xiang Z: A modified protocol for the detection of three different mRNAs with a new-generation in situ hybridization chain reaction on frozen sections. J Mol Histol. 47:511–529. 2016. View Article : Google Scholar : PubMed/NCBI
16	Just L, Timmer M, Tinius J, Stahl F, Deiwick A, Nikkhah G and Bader A: Identification of human cells in brain xenografts and in neural co-cultures of rat by in situ hybridisation with Alu probe. J Neurosci Methods. 126:69–77. 2003. View Article : Google Scholar : PubMed/NCBI
17	Silahtaroglu AN, Nolting D, Dyrskjøt L, Berezikov E, Møller M, Tommerup N and Kauppinen S: Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification. Nat Protoc. 2:2520–2528. 2007. View Article : Google Scholar : PubMed/NCBI
18	Urbanek MO and Krzyzosiak WJ: RNA FISH for detecting expanded repeats in human diseases. Methods. 98:115–123. 2016. View Article : Google Scholar : PubMed/NCBI
19	White AK, Hansen-Lardy L, Brodersen BW, Kelling CL, Hesse RA and Duhamel GE: Enhanced immunohistochemical detection of infectious agents in formalin-fixed, paraffin-embedded tissues following heat-mediated antigen retrieval. J Vet Diagn Invest. 10:214–217. 1998. View Article : Google Scholar : PubMed/NCBI
20	Vicuña L, Strochlic DE, Latremoliere A, Bali KK, Simonetti M, Husainie D, Prokosch S, Riva P, Griffin RS, Njoo C, et al: The serine protease inhibitor SerpinA3N attenuates neuropathic pain by inhibiting T cell-derived leukocyte elastase. Nat Med. 21:518–523. 2015. View Article : Google Scholar : PubMed/NCBI
21	Wang P, Xue Y, Han Y, Lin L, Wu C, Xu S, Jiang Z, Xu J, Liu Q and Cao X: The STAT3-binding long noncoding RNA lnc-DC controls human dendritic cell differentiation. Science. 344:310–313. 2014. View Article : Google Scholar : PubMed/NCBI
22	Faghihi MA, Modarresi F, Khalil AM, Wood DE, Sahagan BG, Morgan TE, Finch CE, St Laurent G III, Kenny PJ and Wahlestedt C: Expression of a noncoding RNA is elevated in Alzheimer's disease and drives rapid feed-forward regulation of beta-secretase. Nat Med. 14:723–730. 2008. View Article : Google Scholar : PubMed/NCBI