Current three-dimensional immunohistochemistry (3D-IHC) methods may reveal key molecular details of biological tissues, but they’re time consuming and limited in depth. In hopes of improving the process and outcome of 3D-IHC, a team of Japanese researchers developed a fast, high-sensitivity method that could support neuroscience and disease diagnostics. Here, Kenta Yamauchi, Assistant Professor, and Hiroyuki Hioki, Professor, at the Juntendo University Graduate School of Medicine, discuss the outcomes of their study.
What inspired this study?
This study was inspired by a desire to visualize targets – such as pathogens and inflammation – within thick brain tissue. Three-dimensional techniques have previously been applied in neurodegenerative research, so we aimed to further investigate the possible applications.
What are the main benefits of using nanobodies fused with peroxidase instead of regular antibodies?
Nanobodies fused with peroxidase (POD-nAbs) offer two key advantages: deeper immunolabeling and more sensitive detection. Their small size (~60 kDa), about 40 percent the weight of conventional IgG antibodies (~150 kDa), allows better penetration into large tissue samples. The peroxidase fusion also enables strong signal amplification through the tyramide signal amplification (TSA) system, FT-GO.
Please tell us about your 3D-IHC method – were there any challenges during its creation?
Our 3D-IHC method – POD-nAb/FT-GO 3D-IHC – relies on three key steps: tissue permeabilization with ScaleA2, target binding by POD-nAbs, and the FT-GO reaction within large tissues. POD-nAbs penetrate tissue rapidly, aided by ScaleA2, and enable strong signal amplification through the TSA reaction.
The biggest challenge we faced was optimizing the IHC conditions – such as solutions, reaction time, and temperature. We tested numerous variations to overcome these hurdles.
How could your new 3D-IHC method help in diagnosing brain diseases like Alzheimer’s or brain tumors?
Our method enables the detection of rare but important structures and molecules. It offers rapid, highly sensitive targeting within large tissue samples, which could be particularly useful for identifying elusive pathogens in brain tissue.
Is this method compatible with common lab samples?
Our 3D-IHC method is compatible with commonly used samples such as formalin-fixed tissue, which we used for all our experiments. However, we have not yet tested its compatibility with paraffin-embedded tissues.
Are enough diagnostic nanobodies currently available to support wide use in pathology?
Currentlavailability of diagnostic nanobodies is limited and not yet sufficient for widespread use in pathology. However, the growing number of publicly available nanobody structures and sequences is expected to expand the repertoire for future applications.
What are your hopes for the future of this technology?
We hope our 3D-IHC technology will be widely adopted in both clinical and experimental research, including studies of diseases beyond the brain, such as cancer and autoimmune conditions. We also hope it inspires others to develop even better 3D-IHC methods, as there is still plenty of room for improvement.
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