Colocalization
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In biomedical studies, the colocalization is commonly understood as the overlap between distinctive labelings in images. This term is usually associated especially with quantitative evaluation of the immunostaining in fluorescence microscopy. On the other hand, the evaluation of the immunolabeling colocalization in the electron microscopy images is still under-investigated and biased by the subjective and non-quantitative interpretation of the image data. We introduce a novel computational technique for quantifying the level of colocalization in pointed patterns. Our approach follows the idea included in the widely used Manders' colocalization coefficients in fluorescence microscopy and represents its counterpart for electron microscopy. In presented methodology, colocalization is understood as the product of the spatial interactions at the single-particle (single-molecule) level. Our approach extends the current significance testing in the immunoelectron microscopy images and establishes the descriptive colocalization coefficients. To demonstrate the performance of the proposed coefficients, we investigated the level of spatial interactions of phosphatidylinositol 4,5-bisphosphate with fibrillarin in nucleoli. We compared the electron microscopy colocalization coefficients with Manders' colocalization coefficients for confocal microscopy and super-resolution structured illumination microscopy. The similar tendency of the values obtained using different colocalization approaches suggests the biological validity of the scientific conclusions. The presented methodology represents a good basis for further development of the quantitative analysis of immunoelectron microscopy data and can be used for studying molecular interactions at the ultrastructural level. Moreover, this methodology can be applied also to the other super-resolution microscopy techniques focused on characterization of discrete pointed structures.
Alzheimer's disease (AD) and sporadic Creutzfeldt-Jakob disease (sCJD) are both characterized by extracellular pathologically conformed aggregates of amyloid proteins-amyloid β-protein (Aβ) and prion protein (PrPSc), respectively. To investigate the potential morphological colocalization of Aβ and PrPSc aggregates, we examined the hippocampal regions (archicortex and neocortex) of 20 subjects with confirmed comorbid AD and sCJD using neurohistopathological analyses, immunohistochemical methods, and confocal fluorescent microscopy. Our data showed that extracellular Aβ and PrPSc aggregates tended to be, in most cases, located separately, and "compound" plaques were relatively rare. We observed PrPSc plaque-like structures in the periphery of the non-compact parts of Aβ plaques, as well as in tau protein-positive dystrophic structures. The AD ABC score according to the NIA-Alzheimer's association guidelines, and prion protein subtype with codon 129 methionine-valine (M/V) polymorphisms in sCJD, while representing key characteristics of these diseases, did not correlate with the morphology of the Aβ/PrPSc co-aggregates. However, our data showed that PrPSc aggregation could dominate during co-aggregation with non-compact Aβ in the periphery of Aβ plaques.
- MeSH
- Alzheimerova nemoc patologie MeSH
- amyloidní beta-protein genetika metabolismus MeSH
- amyloidní plaky patologie MeSH
- Creutzfeldtova-Jakobova nemoc patologie MeSH
- extracelulární prostor chemie MeSH
- jednonukleotidový polymorfismus genetika MeSH
- kodon genetika MeSH
- komorbidita MeSH
- lidé středního věku MeSH
- lidé MeSH
- mozek patologie MeSH
- neurony patologie MeSH
- pilotní projekty MeSH
- proteinové agregáty * MeSH
- proteiny tau metabolismus MeSH
- PrPSc proteiny genetika metabolismus MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- akutní promyelocytární leukemie MeSH
- chromatin MeSH
- hybridizace in situ fluorescenční MeSH
- lidé MeSH
- myši MeSH
- nádorové buňky kultivované MeSH
- progresivní multifokální leukoencefalopatie MeSH
- receptory kyseliny retinové MeSH
- translokace genetická MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
In all eukaryotes, signaling by mitogen-activated protein kinase (MAPK) pathways plays a crucial role in signal transduction during regulation of cell growth, differentiation, proliferation as well as death and stress responses. In this chapter we describe a reliable method to immunolocalize MAPKs in roots of Arabidopsis thaliana by using whole-mount seedling probes. This method relies on quick and efficient chemical fixation, partial cell wall digestion, plasma membrane permeabilization, subsequent antibody incubation, and visualization by high-end confocal laser scanning microscopy (CLSM) performed on whole Arabidopsis seedlings. Protocols are provided for immunofluorescent localization of MPK3, MPK4, and MPK6, representing three major developmentally and stress-regulated MAPKs of Arabidopsis. In addition, protocols for colocalization of these MAPKs with microtubules are also provided.
- MeSH
- Arabidopsis cytologie enzymologie růst a vývoj metabolismus MeSH
- fluorescenční protilátková technika metody MeSH
- konfokální mikroskopie MeSH
- mikrotubuly metabolismus MeSH
- mitogenem aktivované proteinkinasy analýza metabolismus MeSH
- semenáček cytologie enzymologie růst a vývoj MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
