The Whole Picture: Tissue Clearing and 3D Immunodetection

By Alomone Team • June 2024

Have you ever wanted to peer deeper into the world of proteins and cellular structures of your tissues? If so, you’re not alone. The problem is that traditional immunodetection techniques produce 2D flat images since we have to section organs and tissues on account of their opacity. This limits our ability to see how structures exist and interact in three dimensions. But with optical clearing techniques and 3D immunodetection, we can see more than ever – offering deeper insights into protein distributions and interactions. The Power of 3D Immunodetection Picture this: you’re looking at a tissue sample, but instead of a flat section, you’re seeing a comprehensive, detailed view of the entire structure in three dimensions. This is the power of 3D immunodetection, made possible by innovative tissue clearing methods like DISCO and CUBIC. These chemical techniques work by making tissues transparent and allowing visualization of antibodies in the deep layers, which enables mapping out protein distributions like never before. So, what makes optical clearing technique so special?

Deeper visualization: tissue clearing turns tissues transparent, letting antibodies label proteins deep within.
Structural integrity: it preserves tissue architecture, capturing the spatial relationships between structures, cells and proteins
Quantitative analysis: whole-tissue images mean you can more accurately quantify protein expression across the entire sample.

Let’s take a closer look at the scientific contribution of 3D immunodetection in some recent high-impact studies.

Study 1: TRPV1 in Spinal Nerve Compression

In this study (1 ), the researchers investigated the therapeutic effectiveness of harpagoside (HAS), as an epidural medication for spinal nerve compression in rats. By pairing tissue clearing with 3D immunodetection (using Anti-TRPV1 (VR1) Antibody (#ACC-030)), the team could visualize the expression of TRPV1, a protein involved in transmission of pain stimuli, in DRG neurons of rats with induced nerve compression. The 3D view enabled demonstration of TRPV1 distribution across the entire DRG, which reduced significantly following epidural administration of HAS (Figure 1).

Figure 1. Epidural harpagoside (HAS) delivery with pain-relieving effect in rats with lumbar spinal stenosis (LSS). Images stained with NeuN (red) and TRPV1 (green) in cleared DRG tissue from each group. The white boxes indicate magnified regions in the law magnification images.

Study 2: AQP1 and NKCC1 in Cerebrospinal Fluid Production

In research from Denmark (2 ), Li et al. explored the molecular setup of extra-choroidal production of cerebrospinal fluid (CSF) in the central nervous system (CNS) of rodents. Thanks to tissue clearing and 3D immunodetection, the researchers demonstrated expression of AQP-1 and NKCC1 (using Anti-Aquaporin 1 Antibody (#AQP-001) and Anti-NKCC1 (SLC12A2) (extracellular) Antibody (#ANT-071)) in the leptomeningeal vasculature of brain and spinal cord (Figure 2). Both AQP-1 and NKCC1 are proteins involved in CSF production, therefore the leptomeningeal vasculature was proposed to play a role in CSF production.

Figure 2. uDISCO clearance of the intact mouse head depicts the expression of aquaporin 1. a Mouse brain (P60) cleared by uDISCO and immunolabeled for AQP1 (AQP1int, green) reveals the vasculature network in the leptomeninges, including the middle cerebral arteries (MCA, arrows). AQP1+ cells also line the subarachnoid cisterns and the olfactory bulb. b Optical section reveals AQP1+ choroidal epithelial cells and olfactory ensheathing glia cells. c, d Higher magnification images of the areas depicted in b (blue and purple squares) showing AQP1 in the glomerular layer (arrow) and in choroidal epithelial cells (asterisk).

Study 3: HCN4 in Heart Rhythm Disorders

In a study published in Nature Communications (3), the researchers investigated the autonomic innervation associated with regulation of heart rate in mice. Using tissue clearing and 3D immunodetection, visualization and characterization of cardiac neural circuits, with preservation of molecular and cellular architecture, became achievable. Along with anatomical landmarks, 3D expression of the HCN4 protein (using Anti-HCN4 Antibody (#APC-052)) (Figure 3) facilitated anatomical identification of the cardiac conduction system.

Figure 3. AAV-based labeling and tracing of cholinergic neurons on the heart. c To trace cholinergic fibers, presumably from cardiac ganglia, sparse labeling was performed by systemically co-administering ssAAV-PHP.S:TRE-DIO-tdTomato at a high dose (1 × 10¹² vg) and ssAAV-PHP.S:ihSyn1-DIO-tTA at a lower dose (1 × 10¹⁰ vg). Cartoon of the dorsal heart depicting the orientation of images (left). A MIP image of the dorsal atrium with native tdTomato fluorescence (red) and HCN4 staining (green) (middle). Fibers were traced with neuTube and overlaid on a grayscale MIP image (right). Orange fibers coursed along the right atrium (RA) including the sinoatrial (SA) and atrioventricular (AV) nodes and blue fibers along the ventricles. Scale bar is 200 µm.

What’s Next?

3D immunodetection, powered by clever tissue clearing techniques, has the power to give us unparalleled insights into protein distribution within intact tissues. The studies highlighted here validate the role of Alomone Labs antibodies in advancing our knowledge of biology in diverse circumstances. The researchers in the studies above opted for unconjugated primary antibodies, but, in many cases, these are available already conjugated to a fluorescent reporter – Anti-TRPV1 (VR1) (extracellular)-ATTO Fluor-488 Antibody (#ACC-029-AG) and Anti-Aquaporin 1-ATTO Fluor-594 Antibody (#AQP-001-AR), for example. Fluorophore Conjugated Antibodies are making secondary antibodies redundant, cutting down the length of your protocol, and reducing calibration requirements. If you ever need advice on which antibody to pair with a particular tissue clearing method, you can always drop our scientists a message or have a quick call.

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References

1. J. Y. Hong, H. Kim, C. Yeo, J. Lee, W.-J. Jeon, Y. J. Lee, I.-H. Ha, Epidural Injection of Harpagoside for the Recovery of Rats with Lumbar Spinal Stenosis. Cells 12, 2281 (2023). 2. Q. Li, N. N. Aalling, B. Förstera, A. Ertürk, M. Nedergaard, K. Møllgård, A. L. R. Xavier, Aquaporin 1 and the Na+/K+/2Cl− cotransporter 1 are present in the leptomeningeal vasculature of the adult rodent central nervous system. Fluids Barriers CNS 17. 3. P. S. Rajendran, R. C. Challis, C. C. Fowlkes, P. Hanna, J. D. Tompkins, M. C. Jordan, S. Hiyari, B. A. Gabris-Weber, A. Greenbaum, K. Y. Chan, B. E. Deverman, H. Münzberg, J. L. Ardell, G. Salama, V. Gradinaru, K. Shivkumar, Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies. Nat Commun 10, 1944 (2019).