Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

Summary

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Abstract

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Introduction

Axonal mRNA recruitment and local translation enable axons to respond to extracellular stimuli in a temporally and spatially acute manner¹. Intra-axonal protein synthesis is best understood in the context of neurodevelopment where it plays crucial roles in growth cone behavior^2-8, axon pathfinding^9-11 and retrograde signaling^12,13. But far less is known about the functional significance of axonal protein synthesis in post-developmental neurons when axonal mRNA and ribosome levels are greatly reduced^14,15. Mature vertebrate axons have long been thought to be translationally inactive¹⁶. However, recent studies indicate that l....

Protocol

All animal procedures were approved by the IACUC of Columbia University and applicable guidelines for the care and use of laboratory animals were followed. Note: Prepare all buffers used for the ISH procedures in RNase-free or DEPC-treated water. This recommendation is not strictly necessary after ISH has been completed but it is suggested that buffers are still prepared in autoclaved double-distilled water and/or sterilized by filtering.

1. Detection of Atf4 mRNA Localized to Cholinergic Axons in the Adult Mouse Brain using Fluorescence In Situ Hybridization (FISH) Followed by Immunohistochemistry

1. Sample prepa....

Results

A brief summary of the procedures described above is shown in Figure 1.

Optimal detection of Atf4 mRNA granules in cholinergic axons using heat-induced unmasking

When assessing axonal localization of mRNAs, it is critical to be able to identify the axons and to be able to visualize low abundant RNAs. The RNA ISH technology described here enables the detection of RNAs at a single-molecule resolution. Standard protocols using this technology .......

Discussion

In this report we describe the use of a high-resolution ISH technology in the detection of axonally localized Atf4 mRNA. These and previous published studies show that this technology is compatible with antibody-based protein detection in tissues or even whole embryos³³. Importantly, it has been recently used for the detection of Arc mRNA within dendrites of hippocampal neurons³⁴. It can also be combined with histological dyes for tissue staining. Finally, it is suitable for the si.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
custom probe targeting residues 20-1,381 of the mouse Atf4 mRNA (NM_009716)	Advanced Cell Diagnostics	-	probe
custom probe targeting residues 15-1,256 of the human ATF4 mRNA (NM_001675.2)	Advanced Cell Diagnostics	-	probe
negative control probe-DapB	Advanced Cell Diagnostics	310043	probe
positive control probe-mouse Polr2A (optional)	Advanced Cell Diagnostics	312471	probe
positive control probe-human PPIB (optional)	Advanced Cell Diagnostics	313901	probe
RNAscope Fluorescent Multiplex Reagent Kit (for fluorescence detection)	Advanced Cell Diagnostics	320850	in situ hybridization kit
RNAscope 2.0 HD Reagent Kit - BROWN (for chromogenic detection)	Advanced Cell Diagnostics	310035	in situ hybridization kit
Goat polyclonal anti-ChAT antibody	Millipore	AB144P
Luxol Fast Blue-Cresyl Echt Violet Stain Kit	American MasterTech	KTLFB
Clearify clearing agent (xylene substitute)	American MasterTech	CACLEGAL
ProLong Gold mounting  medium with DAPI	Life Technologies	P36935
DPX mounting medium	Sigma	6522