Abstract

Hybridization chain reaction based fluorescence In situhybridization in iDISCO cleared brain tissues

Kumar V, Krolewski DM, Martin B, Akil H, Watson SJ
Society for Neuroscience. 2017.

Fluorescence in situ hybridization (FISH) in intact tissues has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. At present, mainly two fluorescent methods are available for detecting mRNA-probe hybridized complex: 1) Peroxidase catalyzed tyramide amplification method- using Digoxigenin/biotin/FITC labelled oligo/cRNA probes. This method however fails to give uniform signal across the depth in tissues thicker than 300-500 µm which is mainly due to the rapid deposition of reactive tyramide on the superficial layers. Additionally, this method is not fully compatible with CLARITY or iDISCO clearing methods. 2) The Hybridization chain reaction (HCR) method - where binding of single stranded cDNA probes to the target mRNAs initiate chain reactions in which metastable fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers (Choi et al., 2014). Although HCR method has been shown to work with CLARITY, it remains unexplored with clearing methods like iDISCO or CUBIC which depending upon molecular targets and tissue type can yield better volumetric staining and visualization. Hence, we investigated the compatibility of HCR-FISH with different clearing methods- iDISCO and CUBIC along with CLARITY. Following the tissue processing and FISH, slices were imaged on confocal/COLM systems and quantified for the uniformity of cellular labelling and S/N ratio across the tissue depth and region. Based on our preliminary results, iDISCO showed the most uniform labelling of cells and high S/N ratio for FISH in fresh frozen slices thicker than 2 mm, whereas with CLARITY (<400 µm) or CUBIC (<100 µm) failed to show comparable staining. The combination of two simpler methods-HCR and iDISCO provides the best opportunity for high resolution intact tissue FISH. However, generation of functional 50-mer DNA probes flanked by initiator sequence appears to be the most challenging part of this method.

http://www.abstractsonline.com/pp8/#!/4376/presentation/2083