In recent years, Multiplex Immunohistochemistry (mIHC), with its ability to simultaneously detect multiple markers and analyze spatial information, has become an important tool for tumor microenvironment research, immunotherapy evaluation, and spatial pathology studies. However, compared to traditional IHC, the mIHC experimental workflow is more complex. From antibody screening to Panel design, to TSA staining and image analysis, every step can affect the final result.
With the rapid development of tumor microenvironment research, spatial biology, and precision pathological diagnosis, Multiplex Immunofluorescence (mIHC) has become an important tool for tissue sample analysis. Compared with traditional immunohistochemistry (IHC) and conventional immunofluorescence (IF), mIHC enables simultaneous detection of multiple protein markers on the same tissue section, allowing cell phenotype identification, spatial localization analysis, and cell-cell interaction studies.
TRAFs (TNF receptor-associated factors) are a family of multifunctional adaptor proteins that bind to surface receptors and recruit additional proteins to form multiprotein signaling complexes capable of promoting cellular responses. Members of the TRAF family share a common carboxy-terminal "TRAF domain", which mediates interactions with associated proteins; many also contain amino-terminal Zinc/RING finger motifs. The first TRAFs identified, TRAF1 and TRAF2, were found by virtue of their interactions with the cytoplasmic domain of TNF-receptor 2 (TNFRII). The six known TRAFs (TRAF1-6) act as adaptor proteins for a wide range of cell surface receptors and participate in the regulation of cell survival, proliferation, differentiation, and stress responses.
The heterogeneity of CAFs is not driven by genetic variations such as gene mutations or chromosomal abnormalities, but relies on transcriptional reprogramming and epigenetic remodeling, dynamically regulated by multiple factors including tumor cell mutation status, microenvironment signals, spatial localization, and host status. Their plasticity manifests as different CAF subpopulations undergoing phenotypic and functional interconversion with tumor progression and environmental changes, widely participating in the entire process of tumor immune evasion, stromal remodeling, angiogenesis, neural infiltration, and treatment resistance. Therefore, systematically analyzing the layered characteristics of CAF heterogeneity, the regulatory mechanisms of plasticity, and clarifying their role in tumor therapy is of significant academic value and clinical importance for developing precise stromal-targeted anti-tumor strategies and breaking through the bottlenecks of solid tumor treatment.
Traumatic brain injury (TBI) is a worldwide health issue that significantly affects the patient as well as their family. Annual total cost of nonfatal TBI in 2016 was $40.6 billion in the United States. In addition to acute brain injury, even mild cases, can lead to cognitive impairment and long-term psychiatric changes. More long term, TBI patients exhibit lower resilience to neurodegenerative disease-associated pathology. Treatment of TBI is made more difficult due to lack of reliable biomarkers to detect TBI. Several proteins are of interest, which are candidates for measurement in blood after TBI. Glial fibrillary acidic protein (GFAP) is an astrocytic intermediate filament protein. As a cytoskeletal protein, GFAP helps provide structural support to astrocytes, which provide metabolic support to neurons and maintains the blood brain barrier. The number and size of astrocytes, in a process called astrogliosis, is also positively correlated with brain injury. Also abundantly expressed in astrocytes, S100B is commonly used as an astrocytic marker and is positively correlated with TBI. Neurofilament-L (NfL) and tau are part of the neuronal cytoskeleton that provide structure to axons. Axons are covered by a multi-layered membrane called the myelin sheath. Myelin basic protein (MBP) is enriched in myelin and helps maintain its structure. UCHL1 and Enolase-2 are ubiquitin hydrolases and glycolytic enzymes, respectively, that are enriched in neurons. PSD95 is an adaptor protein enriched at postsynaptic sites in neurons. After brain injury, neuron-enriched proteins, as well as proteins that maintain neuronal/axonal integrity, can be measured in the blood, reflecting neuronal damage.
The tricarboxylic acid (TCA) cycle includes various enzymatic reactions that constitute a key part of cellular aerobic respiration. The transport of the glycolytic end product pyruvate into mitochondria and the decarboxylation of pyruvate in the TCA cycle generate energy through oxidative phosphorylation under aerobic conditions. Two inner mitochondrial membrane proteins, mitochondrial pyruvate carrier 1 (MPC1) and mitochondrial pyruvate carrier 2 (MPC2), form a 150 kDa complex and are essential proteins in the facilitated transport of pyruvate into mitochondria . Citrate synthase catalyzes the first and rate-limiting reaction of the TCA cycle. Mitochondrial aconitase 2 (ACO2) catalyzes the conversion of citrate to isocitrate via cis-aconitate. IDH1 and IDH2 are two of the three isocitrate dehydrogenases that catalyze oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). IDH1 functions as a tumor suppressor in the cytoplasm and peroxisomes, whereas IDH2 is in mitochondria and is involved in the TCA cycle. Mutations in IDH2 have also been identified in malignant gliomas. Dihydrolipoamide succinyltransferase (DLST) is a subunit of the α-ketoglutarate dehydrogenase complex, a key enzymatic complex in the TCA cycle. Succinate dehydrogenase subunit A (SDHA) is a component of the TCA cycle and the electron transport chain and is involved in the oxidation of succinate. Fumarase catalyzes the conversion of fumarate to malate. Fumarase deficiency leads to the accumulation of fumarate, an oncometabolite that has been shown to promote epithelial-to-mesenchymal-transition (EMT), a developmental process that has been implicated in oncogenesis.
TSA (Tyramide Signal Amplification) kits are high-sensitivity immunoassay systems developed based on the principle of HRP enzyme-catalyzed fluorescent tyramide molecule deposition. Its core mechanism is to use HRP-labeled secondary antibody to catalyze the activation of fluorescent tyramide, causing it to undergo covalent deposition around the target antigen, thereby achieving local high-density fluorescence enrichment and significantly improving detection sensitivity.
The iterative advancement of spatial pathology technology has completely broken through the limitations of traditional pathological detection, including single markers, weak signals, and missing spatial information, becoming a core supporting technology for tumor microenvironment analysis, disease mechanism research, and precise pathological diagnosis.
