Quantitative analysis of RNAscope staining for c-fos expression in mouse brain tissue as a measure of Neuronal Activation

• Published in: MethodsX
• Main Authors: Cristina Cosi, Michael Millar, Mariana Beltran, Lorcan Sherry, Silvia Gatti-McArthur
• Format: Article
• Language: English
• Published: Elsevier 2021-01-01
• Subjects: Brain c-fos RNAscope in-situ hybridisation
• Online Access: http://www.sciencedirect.com/science/article/pii/S2215016121001412

The expression of c-fos mRNA is an indirect marker of neuronal activity. RNAscope ACD Bio RNAscope (now Biotechne) is a proprietary in-situ mRNA detection technology using branched DNA amplification and z paired probes to deliver a robust and specific assay designed primarily for use on formalin fixed paraffin sections [1]. In the present study we adapted this technology to be used in frozen sections to allow quantitative analysis of c-fos gene expression in different mouse brain regions during neuropharmacology studies. The method was applied by Cosi et al. 2021 [2] and the image analysis is described here in details. • The patented RNAscope (ACD Bio) flourescent in-situ hybridisation technology designed primarily for use on formalin fixed paraffin sections was adapted to be used on frozen section from mouse brain. • We carefully controlled sample preparation and handling to maximise mRNA preservation and used the fluorescent properties of the fast Red substrate combined with fluorescent whole slide scanning and image analysis. • A customized algorithm was set up for image analysis • The method developed permitted the quantitative analysis of c-fos expression in specific brain regions from whole sections.