Labeling
Dotaz
Zobrazit nápovědu
cca 500 s. v různém stránkování
Arterial spin labeling (ASL) je neinvazivní metoda MR využívaná k zobrazení mozkové perfuze. S rostoucími obavami týkajícími se používání kontrastních látek obsahujících gadolinium a zároveň významnými technickými pokroky v implementaci ASL se tato metoda stává středem zájmu různých diagnostických aplikací. V přehledovém článku se zaměřujeme na seznámení čtenářů se základy implementace sekvence ASL v neuroradiologii, diskutujeme optimální parametry skenování pro dosažení nejlepší kvality a přesnosti interpretace dat a poskytujeme přehled diagnostických aplikací v oblastech cerebrovaskulárních onemocnění, neuroonkologie, epilepsie a neurodegenerace. Kromě toho představujeme ukázkové radiologické případy a komentujeme potenciální budoucí vývoj neinvazivních ASL metod.
Arterial spin labeling (ASL) is a non-invasive MRI method used to image cerebral perfusion. Given increasing concerns regarding the use of gadolinium-based contrast agents and significant technical advancements in ASL implementation, the method is gaining attention in various diagnostic applications. This review article aims to familiarize readers with the fundamentals of ASL sequence implementation in neuroradiology, discuss optimal scanning parameters for achieving the highest quality and accuracy in data interpretation, and provide an overview of its diagnostic applications in the areas of cerebrovascular diseases, neuro-oncology, epilepsy, and neurodegeneration. Furthermore, we present illustrative radiological cases and explore the potential future developments of non-invasive ASL techniques.
- MeSH
- arteriae cerebrales diagnostické zobrazování MeSH
- lidé MeSH
- magnetická rezonanční tomografie MeSH
- mozková angiografie MeSH
- mozkový krevní oběh MeSH
- neurozobrazování * metody MeSH
- perfuzní zobrazování * metody MeSH
- spinové značení MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- směrnice pro lékařskou praxi MeSH
Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components, and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo, or real 3D at magnifications ranging from about 10× to 1,000,000×. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualized. In order to identify that protein at high resolution, or to see and quantify its distribution at lower magnifications, samples can be labeled with antibodies. Any surface that can be exposed can potentially be studied in this way. Presented here is a generic method for immunogold labeling for scanning electron microscopy, using two examples of specimens: isolated nuclear envelopes and the cytoskeleton of mammalian culture cells. Various parameters for sample preparation, fixation, immunogold labeling, drying, metal coating, and imaging are discussed so that the best immunogold scanning electron microscopy results can be obtained from different types of specimens.
- MeSH
- antigeny genetika metabolismus MeSH
- barvení a značení metody MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- epoxidové pryskyřice chemie MeSH
- exprese genu MeSH
- fixace tkání metody MeSH
- fixativa chemie MeSH
- formaldehyd chemie MeSH
- imunohistochemie metody MeSH
- jaderný obal metabolismus ultrastruktura MeSH
- koloidní zlato chemie MeSH
- komplex proteinů jaderného póru genetika metabolismus MeSH
- mikroskopie elektronová rastrovací metody MeSH
- mikrotomie MeSH
- oocyty metabolismus ultrastruktura MeSH
- polymery chemie MeSH
- protilátky chemie MeSH
- Xenopus laevis MeSH
- zalévání tkání metody MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The ability to analyze cell division in both spatial and temporal dimensions within an organism is a key requirement in developmental biology. Specialized cell types within individual organs, such as those within shoot and root apical meristems, have often been identified by differences in their rates of proliferation prior to the characterization of distinguishing molecular markers. Replication-dependent labeling of DNA is a widely used method for assaying cell proliferation. The earliest approaches used radioactive labeling with tritiated thymidine, which were later followed by immunodetection of bromodeoxyuridine (BrdU). A major advance in DNA labeling came with the use of 5-ethynyl-2'deoxyuridine (EdU) which has proven to have multiple advantages over BrdU. Here we describe the methodology for analyzing EdU labeling and retention in whole plants and histological sections of Arabidopsis.
- MeSH
- Arabidopsis cytologie ultrastruktura MeSH
- barvení a značení metody MeSH
- deoxyuridin analogy a deriváty analýza MeSH
- DNA rostlinná analýza MeSH
- kořeny rostlin ultrastruktura MeSH
- meristém ultrastruktura MeSH
- proliferace buněk * MeSH
- replikace DNA MeSH
- rostlinné buňky ultrastruktura MeSH
- semenáček ultrastruktura MeSH
- zalévání tkání do parafínu metody MeSH
- Publikační typ
- časopisecké články MeSH
Numerous methods exist for fluorescently labeling proteins either as direct fusion proteins (GFP, RFP, YFP, etc.-attached to the protein of interest) or utilizing accessory proteins to produce fluorescence (SNAP-tag, CLIP-tag), but the significant increase in size that these accompanying proteins add may hinder or impede proper protein folding, cellular localization, or oligomerization. Fluorescently labeling proteins with biarsenical dyes, like FlAsH, circumvents this issue by using a short 6-amino acid tetracysteine motif that binds the membrane-permeable dye and allows visualization of living cells. Here, we report the successful adaptation of FlAsH dye for live-cell imaging of two genera of spirochetes, Leptospira and Borrelia, by labeling inner or outer membrane proteins tagged with tetracysteine motifs. Visualization of labeled spirochetes was possible by fluorescence microscopy and flow cytometry. A subsequent increase in fluorescent signal intensity, including prolonged detection, was achieved by concatenating two copies of the 6-amino acid motif. Overall, we demonstrate several positive attributes of the biarsenical dye system in that the technique is broadly applicable across spirochete genera, the tetracysteine motif is stably retained and does not interfere with protein function throughout the B. burgdorferi infectious cycle, and the membrane-permeable nature of the dyes permits fluorescent detection of proteins in different cellular locations without the need for fixation or permeabilization. Using this method, new avenues of investigation into spirochete morphology and motility, previously inaccessible with large fluorescent proteins, can now be explored.
- MeSH
- bakteriální geny MeSH
- bakteriální proteiny genetika metabolismus MeSH
- barvení a značení * MeSH
- fluorescenční barviva * MeSH
- fluorescenční mikroskopie * MeSH
- lidé MeSH
- membránové proteiny genetika metabolismus MeSH
- myši MeSH
- průtoková cytometrie MeSH
- Spirochaetales cytologie genetika metabolismus MeSH
- spirochetové infekce mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Intramural MeSH
The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.
- MeSH
- barvení a značení MeSH
- biotinylace MeSH
- endocytóza MeSH
- endozomy metabolismus MeSH
- klathrin metabolismus MeSH
- lidé MeSH
- mikroskopie metody MeSH
- nádorové buněčné linie MeSH
- receptor fibroblastových růstových faktorů, typ 4 metabolismus MeSH
- signální transdukce MeSH
- trans-Golgiho síť metabolismus MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
