Highly multiplexed tissue markers

Tissue methods

Multiplexed immunostaining

ImmunoHistoChemistry (IHC) and ImmunoFluorescence (IF) have been the primary tools for imaging protein distribution in cells and tissues for several decades. However, these techniques are associated with several limitations, the major challenge being the capacity to label only one or a few markers per tissue section or cell sample. In both research and clinical settings, it is often advantageous to be able to simultaneously detect and quantify multiple proteins in the same cells or tissue. This need has spurred the rapid development of more multiplexed immunohistochemistry and immunofluorescence (mIHC/IF) methods over the past years. Multiplexing methods enable the study of more complex spatial proteomics, revealing detailed protein distrubution patterns in relation to cell composition, functional states, and cell-cell interactions, even in a tissue context with single-cell resolution. Indeed, mIHC/IF has paved the way for significant advances in areas such as cancer research, where it allows detailed studies of tumour microenvironment and the immune cell landscape, with potential applications in clinical practice for diagnosis, prognosis, and patient stratification.

There are multiple methods for increasing the number of proteins that can be detected on the same tissue section, including cyclic immunofluorescence, tyramide-based mIHC/IF, imaging mass cytometry (IMC) and Multiplexed Ion Beam Imaging (MIBI). In the tissue resource of the HPA, an iterative Tyramide Signal Amplification (TSA)-based method is used for detailed profiling of protein distribution in a growing number of tissues, using a selection of 8 tissue-specific markers that allow for the distinction of more cell types and features within each tissue compared to the standard IHC stainings. In addtion to this effort, another method has been employed to increase the number of proteins detected in tissues. Sections from all of the normal tissue microarrays used for conventional IHC in the tissue resource have been stained with a panel of 46 standardized reference markers, plus nuclear counterstain, using the CO-DEtection by inDExting (CODEX) technology. This provides a highly multiplexed reference map of tissue architecture, immune expression, and cell composition in the HPA tissue microarrays.

mIHC/IF with oligonucleotide-barcoded antibodies

The CODEX method relies on DNA-conjugated antibodies and the cyclic addition and removal of complementary fluorescently labeled DNA probes. All antibodies are added simultaneously to the tissue section, but revealed one by one by adding a fluorescently labeled oligonucleotide complementary (reporter) to the unique oligonucleotide barcode, followed by imaging, stripping, and iterating this cycle (Fig. 1). Each cycle encompasses three different fluorophores, allowing for the repeat process to increase the number of proteins in the same tissue slide up to 60, obtaining images at a single cell resolution. Optimizing the sequence, dilutions and exposure times of the primary conjugated antibodies are crucial steps to avoid carry over of fluorescence signal.

Figure 1. The core principle of highly multiplexed imaging using conjugated antibodies method. 1) All primary barcoded antibodies are stained simultaneously. 2) By cycles of 3 antibodies, the unique sequences hybridize to the complementary fluorescent reporters with the subsequent image acquisition. 3) The reporters are stripped away using a chaotropic solvent. 4) The process is repeated till all the cycles are completed. 5) Once all the antibodies have been revealed, an image postprocessing step is applied to merge all the images in a single file.

A highly multiplexed reference map of human tissues

The results of the highly multiplexed staining of the normal HPA tissue microarrays can be explored in the viewer below (or under each tissue in the Dictionary for normal tissue histology). Use the drop down menu to select the tissue of interest. The number of tissue cores avaliable for each organ varies from two to six, resulting in a total of 121 multi-color images of 43 different tissues. The antibody panel used for all tissues comprised 46 different markers, along nuclear staining, which helps distinguish various tissue structures, immune profiles, and cell types (though not all the markers are present in every tissue). The viewer allow you to select up to 7 markers at a time for display, which can be selected using the drop-down menus associated with each color. The intensity of each marker can be adjusted using the associated slider. The viewer allows you to zoom and pan, and can be opened in full screen mode.

Tissue methods

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