In the field of glioma treatment, immunotherapy development has long been limited by unclear understanding of the tumor immune microenvironment. As the most aggressive malignant tumor in the central nervous system, gliomas, particularly glioblastoma (GBM), have long been labeled as "immune-cold" tumors. Barriers such as the blood-brain barrier hindering immune cell infiltration, high expression of immune checkpoint molecules like PD-L1 by tumor cells, and enrichment of immunosuppressive cells like M2 macrophages contribute to a clinical response rate of less than 15% for therapies like immune checkpoint inhibitors.

Tertiary Lymphoid Structures (TLSs) are ectopic lymphoid aggregates that can develop within or adjacent to tumors, activating anti-tumor immunity. Their maturity status is closely related to patients' immunotherapeutic efficacy and survival, making them highly promising prognostic markers. Opal-TSA multiplex immunofluorescence (mIF) technology, with its high sensitivity and staining flexibility, has become an ideal choice to break through these bottlenecks.

Multiplex immunofluorescence combined with tyramide signal amplification (TSA) technology, based on the principle of enzymatic signal cascade amplification, has broken through the limitations of traditional methods in sensitivity and multi-target detection, and its application research in the field of tumor pathological diagnosis has shown explosive growth in recent years.

In recent years, immune checkpoint inhibitor combination chemotherapy regimens have significantly improved the prognosis of some triple-negative breast cancer (TNBC) patients, but existing biomarkers have obvious limitations. Differences in antibody clone numbers in commercial PD-L1 detection kits lead to inconsistent detection thresholds, and only using "immune cell positivity rate >1%" as the judgment criterion cannot distinguish the cell types expressing PD-L1, resulting in limited accuracy in patient screening with a response rate of only 8%-20%. Therefore, using multiplex immunofluorescence TSA technology to analyze the expression characteristics of PD-L1 on different immune cells has become a key direction for optimizing TNBC patient stratification and treatment decisions.

The cellular composition, functional status, and spatial distribution of the tumor immune microenvironment (TME) are core factors determining tumor progression and immunotherapy efficacy. Precise analysis of TME characteristics has become a key direction in tumor research. However, traditional research tools have significant limitations: single immunohistochemistry (IHC) can only detect a single target, failing to show intercellular interactions; flow cytometry enables multiparametric analysis but requires cell dissociation, losing crucial spatial information; early multiplex fluorescence staining techniques were limited by low signal intensity and poor antibody species compatibility, making them difficult to meet clinical sample analysis needs. Against this backdrop, a multiplex immunofluorescence (mIHC) platform based on tyramide signal amplification (TSA) technology has emerged, providing the possibility for "panoramic" analysis of TME. The team of Viratham Pulsawatdi A published a study titled "A robust multiplex immunofluorescence and digital pathology workflow for the characterisation of the tumour immune microenvironment" in Molecular Oncology. This study took colorectal cancer (CRC) formalin-fixed paraffin-embedded (FFPE) tissues as research objects, and based on the Opal multiplex immunofluorescence platform of TSA technology, constructed and verified a standardized workflow from staining to image analysis, providing a reliable method for precise characterization of tumor immune microenvironment. The following is an in-depth analysis from the aspects of technical essence, experimental logic, core findings and application value.

The fluorescent dyes of the Enklife TSA kits have excellent anti-fade properties, so there is no need to avoid light under fluorescent lamps, and no need to operate in a dark environment during use. However, please avoid direct exposure of the kit to sunlight. Stained slides can usually be stored at 4℃ for 6 to 12 months.

The interaction between cancer and the host immune system mainly occurs in the reactive tissue stroma surrounding tumors, an area known as the Tumor Immune Microenvironment (TIME). The breakthrough clinical application of immune checkpoint inhibitors, particularly targeted therapies against Programmed Death Protein 1 (PD-1, a T-cell co-inhibitory receptor) and Programmed Death Ligand 1 (PD-L1, also known as B7-H1/CD274), has not only propelled cancer immunotherapy into a new era but also made functional analysis of TIME a core focus in both research and clinical settings. Precise characterization of TIME's cellular composition, functional states, and molecular interaction networks is a crucial prerequisite for screening predictive biomarkers for immunotherapy response. Furthermore, deeply clarifying the dual pro-cancer/anti-cancer mechanisms of various immune cells in TIME and their dynamic associations with tumor cells can provide core support for developing novel immunotherapeutic strategies. However, the high heterogeneity of TIME and the challenge of detecting low-abundance immune molecules pose severe challenges to traditional analytical techniques, urgently requiring highly sensitive, multiplex-targeted in situ detection tools to overcome this dilemma.

The Forkhead signaling pathway is a conserved, evolutionarily ancient molecular network centered on Forkhead box (FOX) transcription factors—a family of proteins characterized by a highly conserved "Forkhead" DNA-binding domain (≈100 amino acids) that mediates sequence-specific binding to target gene promoters.

Ferroptosis is regarded as a cell death modality of metabolism. The biochemical mechanisms of ferroptosis involve a complex interaction between oxidative stress, lipid metabolism, and iron homeostasis that results in the peroxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids to produce phospholipid peroxide radicals.

In the field of in situ protein detection in tissues, immunohistochemistry (IHC), conventional immunofluorescence (IF), and multiplex immunofluorescence (TSA) are the three most widely used technologies. All three are based on specific antigen-antibody binding, but they differ fundamentally in detection throughput, sensitivity, and spatial resolution capabilities, which directly determine their applicable scope in scientific research and clinical settings. This article will systematically analyze the differences between the three from three dimensions: technical core, key performance, and applicable scenarios, providing precise references for experimental design and technology selection.