prikaz prve stranice dokumenta Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822)
  Download
PDF 2.68 MB
master's thesis
Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822)
Zagreb: University of Zagreb, Faculty of Science, 2021. urn:nbn:hr:217:014155
Babić, Nency
University of Zagreb
Faculty of Science
Department of Biology

Institutional repository: Repository of the Faculty of Science

Cite this document

Babić, N. (2021). Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822) (Master's thesis). Zagreb: University of Zagreb, Faculty of Science. Retrieved from https://urn.nsk.hr/urn:nbn:hr:217:014155

Babić, Nency. "Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822)." Master's thesis, University of Zagreb, Faculty of Science, 2021. https://urn.nsk.hr/urn:nbn:hr:217:014155

Babić, Nency. "Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822)." Master's thesis, University of Zagreb, Faculty of Science, 2021. https://urn.nsk.hr/urn:nbn:hr:217:014155

Babić, N. (2021). 'Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822)', Master's thesis, University of Zagreb, Faculty of Science, accessed 25 November 2024, https://urn.nsk.hr/urn:nbn:hr:217:014155

Babić N. Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822) [Master's thesis]. Zagreb: University of Zagreb, Faculty of Science; 2021 [cited 2024 November 25] Available at: https://urn.nsk.hr/urn:nbn:hr:217:014155

N. Babić, "Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822)", Master's thesis, University of Zagreb, Faculty of Science, Zagreb, 2021. Available at: https://urn.nsk.hr/urn:nbn:hr:217:014155

Metadata
TitleOptimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822)
Title (english)Optimization of the F-actin protein detection methods in zebrafish, Danio rerio (Hamilton, 1822)
AuthorNency Babić
MentorDean Karaica (mentor)
MentorSofia Ana Blažević (sumentor)
Committee memberSofia Ana Blažević (predsjednik povjerenstva)
Committee memberNada Oršolić (član povjerenstva)
Committee memberAntun Alegro (član povjerenstva)
Committee memberJulija Erhardt (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Science
(Department of Biology)
Zagreb
Defense date and country2021-09-29, Croatia
Scientific / art field,
discipline and subdiscipline		NATURAL SCIENCES
Biology
Abstract
Riba zebrica, Danio rerio (Hamilton, 1822) je u proteklom desetljeću postala važan modelni organizam u biomedicinskim istraživanjima. Zbog tehničkih izazova u pripremi histoloških kriopreparata, većina dosadašnjih istraživanja lokalizacije/ekspresije proteina u zebrici je koristila parafinske i whole-mount preparate. Cilj ovog istraživanja bio je u embrijima/ličinkama ribe zebrice starosti 1.-5. dana poslije oplodnje (broj jedinki/skupini=20): 1) odrediti ključne čimbenike u pripremi tkiva zebrica za izradu kriopreparata očuvane strukture, 2) odrediti optimalni protokol za utvrđivanje lokalizacije/ekspresije fiziološki važnog unutarstaničnog proteina F-aktina upotrebom fluorescentno obojenog faloidina, 3) istražiti starosne razlike u ekspresiji F-aktina u mišićima ribe zebrice, i 4) istražiti upotrebljivost optimiziranog protokola za potrebe imunofluorescencijske analize membranskih proteina zebrice poput Na+/K+-ATPaze. Rezultati su pokazali najbolju kvalitetu kriopreparata debljine 8μm rezanih pri -20 °C nakon inkubacije uzoraka 1 h u 4 % paraformaldehidu (PFA) i smjesi 2,5 % želatine/25 % saharoze. Fluorescencijskom analizom mikroskopskih slika mišića glave i trupa jedinki zebrice utvrđeno je da inkubacija uzoraka 1 h u 4 %PFA omogućuje optimalno vezanje faloidina za protein F-aktin. Daljnjim (imuno)fluorescencijskim analizama utvrđena je starosno-ovisna ekspresija F-aktina u mišićima trupa zebrice te mogućnost upotrebe istog protokola za određivanje lokalizacije/ekspresije membranskog proteina zebrice Na+/K+-ATPaze. Stoga, ovaj optimizirani imuno/histokemijski protokol za pripremu/obradu kriopreparata predstavlja podlogu za istraživanja lokalizacije/ekspresije različitih (ne)membranskih proteina zebrice.
Abstract (english)
Zebrafish, Danio rerio (Hamilton, 1822), became an important model in studying various biomedical issues in the last decade. Tehnical challenges in the preparation of histological cryosections, directed majority of zebrafish protein localization/expression studies to utilize parafin-embedded tissue sections or whole-mount preparations. In this study we used zebrafish embryo/larvae (1-5 days after fertilization;number of individuals/group=20) to: 1) determine key factors for preparation of high-quality zebrafish tissue cryosections, 2) optimize the protocol for localization/expression of physiologically important F-actin protein by utilizing fluorescently-labelled phalloidin, 3) investigate age differences in the expression of F-actin in zebrafish, and 4) determine the use of the optimized protocol for immunofluorescence analysis of membrane proteins like Na+/K+-ATPase. The results revealed that the highest quality of 8μm zebrafish cryosections cut at -20 °C was achieved after 1 h fixation in 4 % paraformaldehyde (PFA) and incubation in 2,5 % gelatine/25 % succrose-mixture. Fluorescence analysis of zebrafish skeletal muscles revealed that 1 h-fixation of samples in 4 %PFA enables optimal binding of phalloidin to F-actin. Further (immuno)fluorescence analyses showed age-dependent expression of F-actin in the zebrafish somitic muscles and usablity of the optimized protocol for localization/expression of Na+/K+-ATPase membrane protein. Thus, this optimized immuno/histochemical protocol provides the basis for bioimaging studies of different (non)membrane zebrafish proteins.
Keywords
Keywords (english)
Languagecroatian
URN:NBNurn:nbn:hr:217:014155
Study programmeTitle: Experimental Biology
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Embargo expiration date: 2023-09-29
Terms of useLicence In copyright icon
Created on2021-10-14 13:16:47