# Novel Chromogens for Immunohistochemistry in Spatial Biology

## TL;DR

This paper introduces new chromogens for immunohistochemistry that enable detailed, multiplex staining of tissues with up to four distinct colors.

## Contribution

The study introduces a novel set of chromogenic substrates for multiplex immunohistochemistry with non-overlapping colors and compatibility with standard practices.

## Key findings

- The new chromogens provide fine cellular detail and non-overlapping spectral profiles.
- Four-color multiplex staining was successfully demonstrated using compatible color combinations.
- Visual analysis without computer assistance can differentiate up to four markers.

## Abstract

Spatial relations between tumor cells and host-infiltrating cells are increasingly important in both basic science and clinical research. In this study, we have tested the feasibility of using standard methods of immunohistochemistry (IHC) in a multiplex staining system using a newly developed set of chromogenic substrates for the peroxidase and alkaline phosphatase enzymes. Using this approach, we have developed a set of chromogens characterized by (1) providing fine cellular detail, (2) non-overlapping spectral profiles, (3) an absence of interactions between chromogens, (4) stability when stored, and (5) compatibility with current standard immunohistochemistry practices. When viewed microscopically under brightfield illumination, the chromogens yielded the following colors: red, black, blue, yellow, brown, and green. By selecting compatible color combinations, we have shown feasibility for four-color multiplex staining. Depending on the particular type of analysis being performed, visual analysis, without the aid of computer-assisted image analysis, was sufficient to differentiate up to four different markers.