HRP-labeling of bacterial extracellular vesicles for transmission electron microscopy imaging

University of Oulu 2022-03-14

Extracellular vesicles (EVs) are small, membrane-bound carriers secreted by all cells. In humans, in addition to body’s own cells, also the bacteria of the microbiota releases bacterial EVs (BEVs). BEVs in human body can be examined from body fluids. For studying BEVs derived from gut microbiota, fecal samples are used. The isolation protocol of BEVs includes ultrafiltration, size-exclusion chromatography, and density gradient ultracentrifugation. For characterization, transmission electron microscopy and nanoparticle tracking analysis are commonly used. Several methods for labeling and imaging EVs have been demonstrated. Yet, none of them are suitable tracking EVs with high resolution imaging. In this thesis, a novel horseradish peroxidase (HRP) -labeling for BEVs for transmission electron microscopy (TEM) is established. For visualizing the HRP, carrying out a diaminobenzidine (DAB) -reaction had to be optimized. With multiple trials of in vitro experiments, a suitable protocol for labeling BEVs was established. As a result of successful HRP-labeling followed by DAB-reaction, BEVs appeared sharply stained on the edges. Further trials in ex vivo conditions were performed by injecting HRP-labeled BEVs to a muscle of a euthanized mouse. Thin sections of mouse muscle tissue in TEM revealed dark-stained clusters of structures resembling BEVs. The outcomes of the ex vivo mouse experiment were promising but will require optimizing in future studies.HRP-leimaus bakteeriperäisille solunulkoisille vesikkeleille läpäisyelektronimikroskooppikuvantamiseen. Tiivistelmä. Kaikki solut erittävät ulkopuolelleen kuljetukseen erikoistuneita pieniä kalvopeitteisiä rakkuloita, solunulkoisia vesikkeleitä. Solunulkoisia vesikkeleitä tuottavat paitsi elimistön omat solut, myös elimistössä elävän mikrobiomin solut. Ihmiselimistön suolistobakteerien tuottamia bakteeriperäisiä solunulkoisia vesikkeleitä tutkitaan ulosteesta, joiden eristämiseksi määrittämiseksi ja tutkimiseksi yhdi

Biologia; info:eu-repo/semantics/masterThesis; info:eu-repo/semantics/publishedVersion

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