Nipam Patel, a developmental biologist at the University of Chicago, and his group study the development of body morphology in arthropods, specifically crustaceans in the Parhyale genus. The team focuses on the role and impact of various homeotic (Hox) genes and their proteins, which encode information to guide the animal’s body plan and the role and development of various limbs.
In Patel’s team’s crustaceans, the researchers have seen these genes have unique functions for appendage patterns that are distinct from the often-studied fly. “It explains how crustaceans can generate so much diversity in their appendage types,” he said. The team explores the role of these genes in body segmentation with a combination of genetic models and fluorescent microscopy.
Because Hox proteins work together to give rise to an animal’s body plan, Patel and his team often want to visualize multiple targets simultaneously in microscopy. However, because most monoclonal antibodies come from mice, this introduces challenges for multiplexing since all the antibodies have the same species origin. Although researchers can overcome this by coupling fluorescent molecules directly to the antibodies, Patel said that those solutions are complicated, time intensive, and reduce the signal amplification that normally helps detect the primary antibody.
As the director of the Marine Biological Laboratory (MBL), though, Patel often communicates with microscopy and antibody companies to trial new equipment and reagents. In 2025, representatives from Molecular Instruments were at the facility and offered Patel samples of a new multiplex kit, called HiFi Encoder, that promised the ability to use multiple antibodies from the same species for fluorescent microscopy studies.
Patel’s research assistant at the MBL, Saanvi Turki, took on the task of testing the kit with his team’s Hox protein antibodies. Because the fly’s Hox localization has been well-characterized, Turki used this as a model for their proof-of-concept trial.
To conduct fluorescent imaging multiplexing with primary antibodies from the same species, researchers select different encoder labels, each specific to a different fluorophore, for each protein they intend to target. Before labeling their sample, the researchers prepare each of their antibodies with its respective encoder tag. The kit uses oligo hairpins to amplify the antibody signal. “The beauty is, it only takes 10 minutes to label the antibody,” Patel said.
However, using multiple colors in microscopy can lead to problems with differentiating the signals from antibodies due to spectral overlap. Because of this, Patel said that it’s important to use instruments that are able to perform spectral imaging, where the microscope collects all of the spectrum data in the image, followed by linear unmixing that involves computational models that determine the source of each signal.
The team conducted their HiFi Encoder trial run on a fly embryo. When the researchers first saw the images, Patel said that they were excited. In the image, despite using five mouse-derived antibodies, the researchers could clearly identify each Hox protein in its anticipated location. “It allows us to do an experiment that otherwise would be extremely difficult to do,” Patel said. Additionally, he said that many of the antibodies they used in this trial are cross-reactive with their model crustacean. “So, it's great. We can do the control experiments in flies. Once we know they work, we can then easily move them across to other organisms,” he said.
Patel recently taught part of MBL’s embryology course, where he introduced the HiFi Encoder technique to the course participants. He said that they had some successes, but also some failures, which is to be expected with an experimental technique.
Overall, though, Patel is excited to apply this new multiplexing method in his work to study everything from appendage development to nervous system function to structural color. “We're still debugging things. We still have some issues, but basically, it's been extremely useful to us because suddenly now a whole bunch of experiments which really weren't very feasible now are easy to do,” he said.
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