دورية أكاديمية
Combining Multiplex Fluorescence in situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections.
| العنوان: | Combining Multiplex Fluorescence in situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections. |
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| المؤلفون: | Dereli AS; Department of Pharmacology, School of Medical Sciences, University of New South Wales., Bailey EJ; Department of Pharmacology, School of Medical Sciences, University of New South Wales., Kumar NN; Department of Pharmacology, School of Medical Sciences, University of New South Wales; natasha.kumar@unsw.edu.au. |
| المصدر: | Journal of visualized experiments : JoVE [J Vis Exp] 2021 Jun 25 (172). Date of Electronic Publication: 2021 Jun 25. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't; Video-Audio Media |
| اللغة: | English |
| بيانات الدورية: | Publisher: MYJoVE Corporation Country of Publication: United States NLM ID: 101313252 Publication Model: Electronic Cited Medium: Internet ISSN: 1940-087X (Electronic) Linking ISSN: 1940087X NLM ISO Abbreviation: J Vis Exp Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: [Boston, Mass. : MYJoVE Corporation, 2006]- |
| مواضيع طبية MeSH: | Brain* , RNA*, Animals ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Mice ; RNA, Messenger |
| مستخلص: | Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A "neurochemical signature" to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section. Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei. Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope. |
| المشرفين على المادة: | 0 (RNA, Messenger) 63231-63-0 (RNA) |
| تواريخ الأحداث: | Date Created: 20210712 Date Completed: 20211015 Latest Revision: 20211015 |
| رمز التحديث: | 20240628 |
| DOI: | 10.3791/61709 |
| PMID: | 34251373 |
| قاعدة البيانات: | MEDLINE |
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