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Zobrazovanie imunohistochemicky značených rezov sferoidov s využitím zlatých nanočastíc pomocou laserovej ablácie a hmotnostnej spektrometrie s indukčne viazanou plazmou

Název česky Zobrazování imunohistchemicky značených řezů sferoidů s využitím zlatých nanočástic pomocí laserové ablace a hmotnostní spektrometrie s indukčně vázaným plazmatem


Druh Konferenční abstrakty
Fakulta / Pracoviště MU

Přírodovědecká fakulta

Popis Spheroids are 3D biological models, where the distribution of proliferating and apoptotic cells contributes to the tumor microenvironment. Meanwhile, a necrotic core is present in the center of the spheroid, outer layers consist mostly of proliferating cells. One of the markers of proliferation is protein Ki-67 expressed by cells that are in active phases of the cell cycle. For imaging of specific proteins in spheroid slides immunohistochemical (IHC) staining with specific primary and fluorophore-labeled secondary antibody followed by fluorescence microscopy analysis is routinely applied. The bridge between primary antibody and a label may be mediated via biotin and streptavidin interaction. Laser ablation inductively coupled plasma mass spectrometry (LA ICP MS) is a technique capable of highly sensitive elemental and isotopic analysis of solid samples. Highly specific qualitative and quantitative analysis is achieved with the use of gold nanoparticles as labels with a defined size. IHC staining of 12 µm slides of spheroids with a diameter of 1 mm derived from colorectal carcinoma cell lines HT-29 is described in this poster. For IHC detection of protein Ki-67 was chosen a system using primary biotinylated antibody and conjugates of streptavidin and gold nanoparticles (60 nm) with the previous blocking of non-specific interactions with various blocking solutions and blocking of endogenous biotin, high specificity binding partner of streptavidin. Samples prepared were analyzed with LA ICP MS with the laser of 2940 nm wavelength. Resulting image comprised of the spatial distribution of nanoparticles, quantity per pixel, as well as their sum per image. The purpose was to develop a method similar to IHC staining with fluorescence microscopy, but with higher sensitivity.
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