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bioRxiv · 10.1101/763920

Optimization and quantitative evaluation of fluorescence in situ hybridization chain reaction in intact fresh-frozen brain tissues

KUMAR, V.·KROLEWSKI, D. M.·HEBDA-BAUER, E. K.·PARSEGIAN, A.·MARTIN, B.·FOLTZ, M.·AKIL, H.·WATSON, S.
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Abstract

Transcript labelling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, it remains relatively unexplored in fresh-frozen brain which provides more flexible utilization than perfused tissue. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labelling between two clearing methods, CLARITY and iDISCO+. We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO+ resulted in better tissue clearing, greater imaging depth and a more uniform labelling of larger samples. Based on these results, we used iDISCO+-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labelling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins. Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatin-expressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods especially iDISCO+ may find broad application in the transcript analysis in rodent studies but its limited use in postmortem human tissues can be improved by further optimizations.

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KUMAR, V., KROLEWSKI, D. M., HEBDA-BAUER, E. K., PARSEGIAN, A., MARTIN, B., FOLTZ, M., AKIL, H., WATSON, S.. 2019-09-10. Optimization and quantitative evaluation of fluorescence in situ hybridization chain reaction in intact fresh-frozen brain tissues. https://doi.org/10.1101/763920

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