Multiplex fluorescence immunohistochemistry (mIHC), also known as tyrosine signal amplification, is a class of enzymatic assays that utilize horseradish peroxidase for high-density in situ labeling of target proteins or nucleic acids.

This method is based on multiple cis-immunostaining techniques with tyrosine signal amplification, allowing the simultaneous detection of multiple targets in a single cell or tissue sample and the comprehensive study of cellular composition, cellular function and cellular interactions.

TSA technology uses an HRP-labeled secondary antibody, which catalyzes the addition of a TSA-derived fluorescent dye to the system, generating an activated fluorescent substrate that covalently binds to the tyrosine on the antigen and covalently binds the signal to the antigen. Afterwards, the non-covalently bound antibody is washed away by thermal repair, and then replaced by the next primary antibody for a second round of incubation with another fluorescein substrate, and so on to achieve multiple labeling.

mIHC achieves in situ multi-target staining of tissues or cells through multiple rounds of staining cycles with the help of different fluorescent dye markers^[2].

Principle: The dye-labeled substrate tyramine (T) molecule is converted to a transiently active intermediate state (T*) by antibody- or probe-immobilized HRP in the oxidizing environment of hydrogen peroxide. The activated intermediate state molecule (T*) rapidly and stably covalently binds to the electron-rich region of the attached protein molecule (tyrosine residue), and the unlabeled tyramine molecule is eluted, thus achieving specific staining for the antigen. Since the conjugated proteins (including HRP, antibody, and target antigen) contain a large number of tyrosine binding sites, the target antigen is enriched with a large number of labeled molecules, which effectively amplifies the signal (Fig. 2).

Differences between IHC and mIHC

1. Conventional immunohistochemistry is a qualitative analysis, and its judgment is mostly based on experience, and its results may vary. While multiple fluorescence immunohistochemistry is a quantitative analysis, its use of software can be accurate analysis of the results of the cells in the tissue.

2. Conventional immunohistochemistry is limited by traditional staining and imaging, with less than 3 targets, and cannot analyze the component information completely. Multi-fluorescence immunohistochemistry can achieve multi-factor co-localization, which can obtain more biological information and requires fewer samples.

Differences between IF and mIHC

Application 1: Tumor Mechanism Exploration

Gang Wei et al. collected perinephric adipose tissue (PAT) and subcutaneous white adipose tissue (WAT) tissue samples from patients with clear cell renal cell carcinoma (ccRCC), and found that the expression of uncoupling protein 1 (UCP1) was higher in adipocytes adjacent to the tumor than in distal adipocytes (one of the typical features of WAT browning is the upregulation of UCP1 expression).

In addition, qRT-PCR, mIHC assay and immunoblotting of tumor cells showed that the adipocyte-promoted up-regulation of UCP1 levels was suppressed in tumors co-injected with BAC-shPgc1a or BAC-shUcp1-associated tumors (Fig 4).

Further studies revealed that ccRCC cells secrete the parathyroid hormone-related protein PTHrP to promote PAT browning via PKA activation, and PAT-mediated thermogenesis leads to the release of excess lactate to enhance growth, invasion and metastatic coloration of ccRCC^[3].

Application 2: Identification of immunosuppressive tumor microenvironments

Halse, H et al. used mIHC to pinpoint immune subpopulations in metastatic melanoma, and the data quantified the number of immune subpopulations within the tumor (Intra-tumor, IT) and the tumor stroma (Peri-tumoral, stroma), as well as the distances of immune subpopulations from (Tumor margin, TM).

As shown, the precise location of immune subpopulations in metastatic melanoma was analyzed using mIHC in specific tumor regions to observe the expression of different immune subpopulations within the tumor. The results showed that for MelTIL026 (pelvic lymph node) sections of melanoma, the majority of T cells were CD4⁺ T cells and were prominent in the tumor margin (TM) and mesenchymal regions (S), with lower numbers of CD4⁺ and CD8⁺ T cells in the intratumor (IT) region.