Rhapontin

Sirius Red or picrosirius red staining is a histochemical method for visualizing collagen-rich extracellular matrix in tissue sections, and collagen fibers are detected as red-stained structures under bright-field microscopy with enhanced birefringence under polarized light. Picrosirius red is useful for assessing total collagen organization, distribution, and fibrosis burden, but polarized color should not be interpreted as a definitive collagen type I versus type III readout because color is affected by fiber orientation, thickness, and packing.

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

Western blotting (WB) is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics for identifying and quantifying target proteins. It combines gel electrophoresis with immunoassay, enabling researchers to analyze protein expression, post-translational modifications, and molecular weight.

ECM-embedded organoid dome culture embeds epithelial stem cells, crypts, organoid fragments, or tumor-derived epithelial cells in a basement-membrane-like hydrogel such as Matrigel, allowing 3D growth, self-organization, lumen formation, budding or cystic morphogenesis, and lineage maintenance under defined niche-factor-containing medium. The primary readouts are organoid establishment efficiency, growth, morphology, passaging capacity, lineage-marker expression, and, when fluorescently labeled lines are used, microscopy- or flow-cytometry-based quantification of population behavior in 3D culture.

Masson’s Trichrome (collagen/trichrome staining) is a histological technique that differentially stains tissue compartments using sequential acidic dyes to distinguish collagen from muscle and cytoplasmic components based on dye affinity and tissue permeability differences, enabling visualization of fibrosis and connective tissue architecture in histological sections. The classical formulation typically uses Weigert's iron hematoxylin for nuclear staining, Biebrich scarlet-acid fuchsin for cytoplasm and muscle, and aniline blue (or light green variants) for collagen, producing a characteristic blue/green collagen signal contrasted against red cytoplasm and dark nuclei. The staining principle relies on selective displacement of smaller dye molecules by larger anionic dyes in collagen-rich regions under controlled acidified conditions, which enhances collagen-specific dye retention. This property makes the method widely used for fibrosis assessment in organs such as heart, liver, lung, a

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

Matrigel Transwell/Boyden chamber invasion assay measures the ability of cells to degrade or traverse an extracellular matrix-coated porous membrane and move from an upper chamber toward a chemoattractant in a lower chamber. Invasion is distinguished from migration by coating the membrane with Matrigel or basement membrane matrix; uncoated inserts measure migration, while coated inserts require cells to cross an ECM barrier before reaching the underside of the membrane.

Recombinant protein expression in Escherichia coli followed by purification of a His-tagged soluble protein by immobilized metal affinity chromatography (IMAC), with optional MBP fusion and TEV tag removal when the construct includes these elements. The biological readout is production of the encoded target protein, detected as an inducible band at the expected molecular mass by SDS-PAGE and quantified by total protein assay or chromatographic absorbance; the purification readout is enrichment of the target protein in elution fractions after selective binding of polyhistidine residues to immobilized Ni2+/metal-chelate resin and elution by imidazole-containing buffer. Expression is driven by an inducible bacterial expression system, commonly T7/lac-based, in which IPTG or lactose/auto-induction activates transcription and translation of the cloned gene; lower induction temperature, lower inducer concentration, induction timing, and solubility-enhancing fusion tags can influence the frac

Fibrosis and collagen morphometry is based on the quantitative visualization of fibrillar collagen deposition in tissue sections using histochemical stains such as Sirius Red (Picrosirius Red) or Masson's trichrome, followed by image-based or polarization-enhanced analysis to estimate collagen proportional area as a surrogate of extracellular matrix accumulation during fibrotic remodeling. Sirius Red combined with polarized light microscopy enhances detection of collagen fibers due to birefringence properties, enabling more specific visualization of collagen type I and III fibrils compared to conventional bright-field histology, while whole-section or region-restricted digital morphometry reduces field-selection bias in fibrosis assessment. Alternative quantitative approaches include second harmonic generation (SHG) and two-photon excited fluorescence microscopy, which enable label-free detection of fibrillar collagen and have been validated against histological staining and biochemica

Dextran sulfate sodium (DSS)-induced colitis is generated by administering DSS in mouse drinking water, producing epithelial injury, barrier disruption, weight loss, diarrhea, fecal blood, colon shortening, histologic mucosal damage, and inflammatory mediator changes; the model is mainly used to study acute or chronic intestinal inflammation resembling selected features of ulcerative colitis. DSS injury is interpreted through clinical and tissue readouts rather than a single molecular endpoint: daily body weight, stool consistency, and bleeding are combined into a disease activity index, while colon length, histology, cytokines, myeloperoxidase activity, intestinal permeability, and tight-junction markers provide complementary measures of inflammation and barrier damage.

TNBS-induced colitis is produced by intrarectal delivery of 2,4,6-trinitrobenzene sulfonic acid in ethanol, where ethanol disrupts the mucosal barrier and TNBS haptenates colonic proteins, generating immune-mediated colonic inflammation with weight loss, diarrhea, ulceration, transmural injury, inflammatory-cell infiltration, and cytokine responses. The model is used as an experimental intestinal inflammation model with Crohn’s disease–like features, especially when Th1-type responses, IL-12–dependent inflammation, chronic relapsing inflammation, or fibrosis-related endpoints are studied.

The Matrigel/ECM Transwell invasion assay measures the ability of cells to move toward a chemoattractant while crossing an extracellular-matrix barrier placed on a porous membrane; therefore, the readout reflects both chemotactic motility and matrix invasion rather than migration alone. Matrigel is a basement-membrane-rich matrix derived from Engelbreth-Holm-Swarm mouse sarcoma and has been used as a reconstituted basement membrane barrier in chemoinvasion assays. The assay readout is generated by quantifying cells that reach the underside of the insert membrane or lower compartment after incubation, commonly by staining and counting invaded cells or by fluorescence-based quantification.

Pyroptosis is a lytic inflammatory cell-death pathway executed by gasdermin pores, most classically through inflammasome-mediated activation of caspase-1, cleavage of gasdermin D, membrane pore formation, LDH release, and secretion of IL-1β and IL-18. The canonical pathway is commonly modeled by priming cells with an inflammatory signal such as LPS to induce pro-IL-1β and inflammasome components, followed by an activation signal such as ATP or nigericin to activate NLRP3, ASC speck formation, caspase-1 cleavage, GSDMD cleavage, cytokine release, and pyroptotic membrane rupture. The non-canonical pathway is triggered when cytosolic LPS activates mouse caspase-11 or human caspase-4/5, leading to GSDMD cleavage and pyroptosis, and this can secondarily activate NLRP3-dependent IL-1β release. Pyroptosis is linked to inflammatory injury, infection, cancer, liver disease, ocular disease, placental inflammation, and other disease phenotypes, but unresolved questions include which gasdermin fam