CN016

Multiplex immunofluorescence IHC detects multiple protein biomarkers in one tissue section by sequential antibody staining, HRP-mediated tyramide fluorophore deposition, heat-mediated antibody stripping, nuclear counterstaining, multispectral imaging, spectral unmixing, and digital cell phenotyping; TSA deposits fluorophore near the antigen so the fluorescence signal remains after primary and secondary antibodies are removed, enabling repeated staining cycles, including with antibodies from the same host species. Classic FFPE tumor immune-profiling applications use panels such as CD3, CD8, CD68/CD163, FOXP3, PD-1, PD-L1, pancytokeratin, Ki67, and DAPI to identify tumor cells, immune-cell subsets, checkpoint-marker expression, co-expression phenotypes, cell density, and spatial relationships in the tumor microenvironment.

This protocol assesses in vitro neurotoxicity by combining neuronal viability, mitochondrial/metabolic activity, neurite outgrowth, and optional neuronal network function readouts. Calcein-AM or resazurin/PrestoBlue readouts estimate viable or metabolically active cells; βIII-tubulin immunofluorescence detects neuronal morphology and neurite networks; TMRE detects mitochondrial membrane potential; and MEA recordings detect functional changes in neuronal network activity.

Lipopolysaccharide (LPS)-induced endotoxemia is a widely used in vivo model of acute systemic inflammation in which LPS, a Gram-negative bacterial endotoxin, activates innate immune signaling primarily through TLR4, leading to rapid and transient induction of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in circulation and tissues. This cytokine surge is commonly used as a measurable readout of systemic inflammatory activation and immune dysregulation, and is typically assessed within hours after intraperitoneal LPS administration in mouse models of endotoxemia. The model captures key features of systemic inflammatory response syndrome, including cytokine release, immune cell activation, and downstream tissue responses, and has been used to evaluate anti-inflammatory interventions such as cytokine modulation, lipid mediators, and immune cell-targeting therapies.

Multiplex immunohistochemistry (mIHC), also known as tyramide dignal amplification (TSA), is an enzymatic detection method that uses horseradish peroxidase (HRP) to perform high-density in-situ labeling of target proteins or nucleic acids.

The NLRP3 inflammasome is a cytosolic innate immune signaling platform that integrates priming signals and danger-signal activation to promote caspase-1 activation, maturation of IL-1β and IL-18, and gasdermin D-mediated pyroptotic cell death. The core experimental logic is to determine whether inflammatory disease phenotypes are driven by increased NLRP3 expression, ASC-containing inflammasome assembly, caspase-1 cleavage, GSDMD cleavage, and extracellular release of IL-1β/IL-18 rather than by nonspecific cell injury alone. The pathway is strongly linked to inflammation-related disease phenotypes because monosodium urate crystals activate NALP3/NLRP3 inflammasome signaling in gout-like crystal inflammation, cholesterol crystals activate NLRP3 inflammasomes in atherogenesis models, and DSS-induced intestinal inflammation has been reported to involve NLRP3 inflammasome activity. However, experimental colitis studies also show context-dependent protective effects of NLRP3 inflammasome co

Cancer immunology studies how the immune system recognizes, suppresses, edits, or fails to eliminate malignant cells through tumor antigen release, antigen presentation, T-cell priming, immune trafficking, tumor-cell killing, and feedback inhibition in the tumor microenvironment. The cancer-immunity cycle links tumor antigenicity, dendritic-cell priming, CD8+ T-cell infiltration, cytotoxic function, and immune-checkpoint regulation to tumor rejection or immune escape. Immune-checkpoint pathways such as PD-1/PD-L1 and CTLA-4 suppress antitumor T-cell activity and can be therapeutically blocked, but many tumors remain resistant because of poor antigen presentation, weak T-cell infiltration, suppressive myeloid cells, regulatory T cells, and tumor-intrinsic immune-exclusion programs. Unresolved questions include which immune-cell states predict response, how tumor-intrinsic pathways exclude immune cells, how myeloid suppression limits checkpoint blockade, and which combination strategies