In situ host-guest chemistry for three-dimensional histology, or more simply, INSIHGT. We know, we know, “Another acronym!”, but this one’s a technique that takes the pain and inconsistencies of 3D staining and fixes them. And there’s no clearing – just chemistry. The science? Ingenious. The results? Solid. The headaches it solves? Many!
The Science: Weakly Coordinating Superchaotropes and Host-Guest Chemistry
INSIHGT is built around two key players: weakly coordinating superchaotropes (WCS) and host-guest chemistry involving γ-cyclodextrins (1). Let’s give you a quick intro to what these mean.
- Chaotropes are molecules that disrupt structured interactions, like those in proteins or between antibodies and antigens.
- Superchaotropes are essentially ‘next-level’ chaotropes. They’re really good at weakening these interactions without destroying the overall integrity of the system.
- Host-guest chemistry is an interaction where one molecule (the “host”) forms a reversible, non-covalent complex with another molecule (the “guest”). Think of it like a lock and key that can click together temporarily but easily separate when needed.
- γ-cyclodextrins are the host molecules in this case. They’re ring-shaped sugars that can trap smaller molecules (guests) inside their structure. In INSIHGT, γ-cyclodextrins help stabilize the disrupted system by re-binding the antibody to its target after the superchaotrope has done its job.
And here’s how it works. WCS, like closo-dodecaborates, temporarily loosen up antibody-antigen interactions without destroying your sample. This step allows the antibodies to diffuse deep into the tissue. Once infiltrated, γ-cyclodextrins help the antibodies reconnect with their targets to re-establish the bonds you disrupted earlier through host-guest chemistry.
With INSIHGT, you simply incubate your formaldehyde-fixed tissue with WCS plus some PBS with antibodies, then add in cyclodextrin and that’s it – both at room temperature with no need for fancy, proprietary equipment, just some solid chemical wizardry. The result? Deep, uniform 3D staining that even works in thick tissues – and all without clearing.
The Challenges INSIHGT Conquers
3D histology has always been a bit of a challenge, to say the least. Probes struggle to reach the inner layers of a sample, leaving deeper structures poorly stained. Surface markers look great, but deeper signals? Not so much. Clearing protocols are long and often need endless optimization to get a maybe-usable sample. And there’s typically a major dependence of equipment that many labs simply don’t have access to.
INSIHGT addresses all of this. Penetration is now much smoother, staining is uniform, and the process is fast. Plus, it’s affordable and accessible to labs without unlimited budgets.
INSIHGT Delivers the Data
If you’re skeptical, the data speaks for itself. In Figure 1, INSIHGT is stacked against other 3D methods, and it showed deep tissue penetration with minimal loss of signal. The contrast and uniformity were consistently sharp, even in thick tissue. Later in the paper they also go on to run high-throughput whole-organ deep immunostaining, where they used our Anti-Aquaporin 5 Antibody (#AQP-005).
Figure 1. Homogeneous and deep staining with INSIHGT
. A) INSIHGT disrupts antibody-antigen interactions using superchaotropes for deep probe penetration, then restores binding with γ-cyclodextrins to achieve uniform staining. B) Benchmarking shows INSIHGT produces clearer, more consistent deep tissue staining than other buffers, with strong bulk-staining (magenta) and minimal cut-staining (green). C) INSIHGT minimizes signal decay with depth, closely matching ideal staining performance (gray line). D) INSIHGT delivers accurate 3D immunostaining, with strong correlations between its signals and reference intensities. E) INSIHGT processes whole brains faster than other methods and could work even quicker with secondary antibody fragments. Full legend at https://www.biorxiv.org/content/10.1101/2024.05.24.595771v2.full
But the best bit comes in Figure 2. Here, INSIHGT tackled human brain samples – 3.5mm-thick human cerebellum samples to be precise – and it delivers results that rival (or surpass) more cumbersome techniques.
Figure 2. INSIHGT enables non-destructive characterization and analysis of human clinical samples
A) INSIHGT stains a 3.5 mm-thick human cerebellum for glial filaments (GFAP), neurofilaments (NF-H), and blood vessels, revealing detailed structures. B-C) INSIHGT visualizes the orientation and coherence of neurofilaments (B) and glial filaments (C) using structure tensor analysis. D) Post-hoc confocal microscopy captures high-resolution details within selected regions of the cerebellum. E) INSIHGT identifies and traces neurofilament inclusions, showing their looping connections to the Purkinje layer and occasionally to other inclusion bodies. F) Manual tracing of 1,078 neurofilament inclusions demonstrates z-depth color coding for comprehensive 3D analysis. Full legend at https://www.biorxiv.org/content/10.1101/2024.05.24.595771v2.full
Making 3D Models for All
3D model shouldn’t be a luxury – they’re essential for understanding spatially complex systems and quite often, the traditional flat, 2D slice approach just doesn’t cut it. And while INSIHGT isn’t perfect, it’s a big step forward. It’s faster, simpler, and more robust than many existing methods. For researchers who’ve hesitated to dive into 3D histology because of the challenges, this could be the turning point.
At Alomone Labs, we’ve long believed that 3D biology is the future. It’s why we focus on research tools like conjugated antibodies and toxins tailored for these applications. INSIHGT feels like it fits in perfectly with this: an innovation that embraces the intricacies of 3D biology while staying practical for everyday use.
Alomone Labs products mentioned in this blog:
| Anti-Aquaporin 5 Antibody (#AQP-005) |
Reference
- N. Yau, J. T. S. Hung, R. A. A. Campbell, T. C. Y. Wong, B. Huang, B. T. Y. Wong, N. K. N. Chow, L. Zhang, E. P. L. Tsoi, Y. Tan, J. J. X. Li, Y. K. Wing, H. M. Lai, INSIHGT: An accessible multi-scale, multi-modal 3D spatial biology platform. bioRxiv [Preprint] (2024). https://doi.org/10.1101/2024.05.24.595771.
Print this post
Share this post