Precise analysis of the tumor microenvironment is the core foundation for cancer diagnosis, prognosis assessment, and treatment decision-making. However, traditional tissue imaging techniques have obvious limitations: single immunofluorescence (IF) imaging can detect multiple molecular markers but lacks histological context, making it difficult to accurately localize cell types and distributions. Hematoxylin-Eosin (H&E) staining, as the gold standard for pathological diagnosis, clearly presents tissue morphology but cannot provide molecular-level immune characteristic information. Existing multiplex imaging techniques either have complex processes and are time-consuming, or have issues such as fluorescence crosstalk and insufficient reagent stability, making it difficult to achieve simultaneous precise detection of molecular markers + tissue morphology.

Housekeeping proteins perform numerous basic functions within the cell and are constitutively expressed at high levels in a variety of tissues and cell types. Western blot analysis commonly uses housekeeping proteins such as β-actin, COX IV, GAPDH, histone H3 and the α- and β-tubulins as loading controls.

Jak (Janus Kinase) and Stat (signal transducer and activator of transcription) proteins are utilized by receptors for a wide varity of ligands including cytokines, hormones, growth factors, and neurotransmitters. Jaks and Stats play important roles in oncogenesis, tumor progression, angiogenesis, cell motility, immune responses, and stem cell differentiation. Therefore, regulation of Jak/Stat signaling is crucial to prevent aberrant signaling which can lead to disease progression.

The four JAK family members, Jak1, Jak2, Jak3 and Tyk2, range in size from 120 to 140 kDa, and except for Jak3 (leukocyte-JAK), are expressed in most tissues (reviewed in).

Interferon regulatory factors (IRFs) comprise a family of transcription factors that function within the Jak/Stat pathway to regulate interferon (IFN) and IFN-inducible gene expression in response to viral infection.

Interleukin-1 (IL-1) receptor-associated kinase (IRAK) is a serine/threonine-specific kinase that can be copreciInterleukin-1 (IL-1) pitated in an IL-1-inducible manner with the IL-1 receptor. The mammalian family of IRAK molecules contains four members (IRAK1, IRAK2, IRAK3/IRAK-M, and IRAK4).

Integrins are α/β heterodimeric cell surface receptors that play a pivotal role in cell adhesion and migration, as well as in growth and survival.

Apoptosis is a regulated physiological process leading to cell death. Caspases, a family of cysteine acid proteases, are central regulators of apoptosis. Initiator caspases (including 2, 8, 9, 10 and 12) are closely coupled to proapoptotic signals, which include FasL, TNF-α, and DNA damage. Once activated, these caspases cleave and activate downstream effector caspases (including 3, 6 and 7), which in turn cleave cytoskeletal and nuclear proteins such as PARP, α-fodrin, DFF and lamin A; inducing apoptosis.

Increased numbers of tumor-infiltrating lymphocytes (TILs) are closely associated with favorable clinical outcomes in various human cancers. These cells were initially identified and defined by pathologists in hematoxylin-eosin (H&E) stained sections, and their distribution and quantity are important prognostic indicators for melanoma.

The immune system has the capability to detect and eliminate tumor cells, most notably through cytotoxic CD8+ T cell recognition of tumor antigens presented on the surface of tumor cells by MHC class I molecules. However, anti-tumor immune cells can be subject to various immunosuppressive mechanisms in the tumor microenvironment (TME).