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. 2024 May 29;13(11):936.
doi: 10.3390/cells13110936.

Novel Chromogens for Immunohistochemistry in Spatial Biology

Bipin Gupta  1 , George Yang  1 , Marc Key  2
Affiliations

Novel Chromogens for Immunohistochemistry in Spatial Biology

Bipin Gupta et al. Cells. .

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.

Keywords: brightfield; chromogens; immunohistochemistry; multiplex; spatial biology.

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Conflict of interest statement

The authors were employed by, or received funding from, Diagnostic BioSystems, Inc.

Figures

Figure 1
Figure 1
Optimization of chromogens on tonsil tissue. Tonsil tissue stained with chromogens (100×). (A) Pan-cytokeratin (AE1/AE3) with green-HRP, (B) pan-cytokeratin (AE1/AE3) with blue-HRP, (C) pan-cytokeratin (AE1/AE3) with yellow-HRP, (D) pan-cytokeratin (AE1/AE3) with red-AP, (E) B-cell (CD20) with yellow-HRP, (F) B-cell (CD20) with red-AP, (G) macrophage (CD163) with DAB, (H) pan-cytokeratin (AE1/AE3) with yellow-HRP and B-cell (CD20) with blue-HRP, and (I) pan-cytokeratin (AE1/AE3) with green-HRP and B-cell (CD20) with yellow-HRP.
Figure 2
Figure 2
Four-plex stain on colorectal carcinoma using chromogens for HRP. Colorectal carcinoma (100×). Pan-cytokeratin (AE1/AE3) stained with yellow-HRP. Macrophage (CD163) stained with DAB. B-cell (CD20) stained with blue-HRP. Proliferation marker (Ki67) stained with green-HRP. Box A shows an area of the tumor at its original location (smaller Box A) and a higher magnification of the selected area (larger Box A).
Figure 3
Figure 3
Four-plex stain on colorectal carcinoma using chromogens for AP and HRP. Colorectal carcinoma (100×). Pan-cytokeratin (AE1/AE3) stained with red-AP. Macrophage (CD163) stained with DAB. B-cell (CD20) stained with blue-HRP. Proliferation marker (Ki67) stained with green-HRP. Box A shows an area of predominantly B-cells (CD20). Box B shows an area of predominantly macrophages (CD163). Box C shows an area of predominantly tumor cells (AE1/AE3) that are also positive for the proliferation marker (Ki67). Small boxes A, B, and C show the tumor areas in their original locations, and larger boxes A, B, and C show a higher magnification of the same corresponding areas.
Figure 4
Figure 4
Four-plex stain of colorectal carcinoma showing inflammatory cells. Colorectal carcinoma (100×) showing an area of tumor stroma with an extensive inflammatory cell infiltrate. Tumor cells are stained with red-AP for pan-cytokeratin (AE1/AE3) and green-HRP for the proliferation marker (Ki67). Macrophages (CD68) are stained with DAB and B-cells (CD79a) are stained with blue-HRP. The small boxes A and B show the tumor areas at their original locations and the large boxes A and B show the same corresponding areas at a higher magnification.
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