Click Chemistry for Visualization of Newly Synthesized RNA and Antibody Labeling on Ultrathin Tissue Sections

Click chemistry consists of synthesis reactions that are rapid, selective, high-yield, and have no side reactions or byproducts [1]. Because click reactions are biorthogonal and occur under mild conditions, they are compatible with biological systems and can be combined with metabolic labeling of nucleic acids [2] and proteins [3]. For metabolic labeling of mRNA, living cells or tissue are treated with a uridine analog bearing a functional group that can be used to capture labeled mRNA or to visualize its distribution in situ [4]. Detection of the functional group is typically performed with an antibody, but click chemistry provides better sensitivity, specificity, and spatial resolution for microscopy [2]. Metabolic labeling of mRNA is of particular interest in studying plastic changes in brain tissue, which involve complex gene expression cascades and precise spatiotemporal regulation of subcellular mRNA localization [5]. Many open questions remain about the dynamics of transcription and translation in the living brain, especially with respect to the kinetics of mRNA trafficking within neuronal processes [5,6].

Click chemistry-based mRNA labeling can be performed with 5-ethynyl uridine (5-EU), an alkyne-functionalized uridine. EU-containing mRNA can then be visualized by reacting it with an azide-conjugated tag, such as a fluorophore or biotin, via a copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction [2,4]. This approach has been used to visualize EU-labeled mRNA in brain tissue by fluorescence microscopy in mice [7] and zebrafish [8], but the density of brain tissue precludes fluorescence imaging of labeled mRNA in neuronal processes, which could only be done in isolated neurons in vitro [8]. EU-labeled mRNA has been detected in axons in vivo by pre-embedding electron microscopy (EM) [9], but this approach is non-quantitative, inefficient for examining large areas, and is difficult to combine with other detection methods such as immunolabeling.

Ultrathin sections of resin-embedded tissue provide greatly enhanced spatial resolution for fluorescence microscopy relative to sections of standard thickness while offering more flexibility in imaging scale and multiplexed labeling. To optimize a protocol for visualizing EU on ultrathin sections of vertebrate brain, we performed a single stereotactic infusion of EU into the lateral amygdala of adult rats via a cannula. After allowing 30 min for the EU to be incorporated, the brains were fixed by transcardial perfusion of aldehyde fixatives and vibratome sections containing the amygdala were embedded in methacrylate resin. No crosslinker was included in the resin so that sections could be easily de-plasticized before labeling. The two publications that detected EU in mouse brain relied on commercial kits, which are expensive and difficult to troubleshoot. To avoid the use of kits, we adapted a protocol used to label EU in bacteria [10], which detected EU in both fresh fixed (Figure 1a) and ultrathin resin embedded sections (Figure 1b). By following click chemistry with immunofluorescence on ultrathin sections, we were able to colocalize newly synthesized mRNA with the dendritic marker MAP2 (Figure 1c) and the axonal marker SMI312 (Figure 1d).

Fig. 1.

Labeling of newly synthesized RNA using click chemistry on brain sections. a. Fresh free-floating 50μm vibratome brain section. b. Resin embedded 100nm brain section. Intense labeling was present in the nucleolus (white arrowhead) with diffuse labeling throughout the nucleus of the cells (white arrow). Scale bar: 10μm.

In addition to metabolic labeling, click chemistry potentially has an advantage in signal amplification for immunohistochemistry. To increase the number of probe molecules (e.g. fluorophores) that detect each primary antibody molecule, signal amplification methods strategies rely either on multiple layers of labeled affinity probes, such as streptavidin and biotin, or on enzyme-catalyzed reporter deposition. Both strategies degrade spatial resolution, the former because of the bulk of the reagents and the latter because of the diffusion of the reporter. Click chemistry directly conjugates probes to target functional groups, so spatial resolution is determined by the functional groups alone. We developed a strategy for immunofluorescence on ultrathin sections in which a primary antibody was detected first with a biotinylated secondary antibody, followed by streptavidin. Typically, detection would be limited to fluorophores directly conjugated to streptavidin. Streptavidin has four biotin-binding sites, however, so to take advantage of these we applied a biotin conjugated to a click chemistry functional group. In this case we did not use the popular CuAAC reaction [2], but instead chose an orthogonal click chemistry approach, the copper-free inverse electron demand Diels-Alder (IEDDA) reaction or copper-free click chemistry [11]. In this case the biotin was functionalized with tetrazine, which was detected by click conjugation to TCO-Cy5 (Figure 2). We expect that our approach will be useful for mRNA and protein detection in tissue types other than brain.

Fig. 2.

Tetrazine ligation with TCO to protein labeling. Parvalbumin (PV) labeling using copper-free click chemistry reaction (IEDDA) on a 100nm resin section. Scale bar: 10μm.