Optimizacija metoda detekcije proteina F-aktina u zebrici, Danio rerio (Hamilton, 1822)

master's thesis

• Title (english): Optimization of the F-actin protein detection methods in zebrafish, Danio rerio (Hamilton, 1822)
• Author: Nency Babić
• Mentor: Dean Karaica (mentor)
• Mentor: Sofia Ana Blažević (sumentor)
• Committee member: Sofia Ana Blažević (predsjednik povjerenstva)
• Committee member: Nada Oršolić (član povjerenstva)
• Committee member: Antun Alegro (član povjerenstva)
• Committee member: Julija Erhardt (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2021-09-29, 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: kriopreparati; (imuno)fluorescencijska analiza; aktin; natrij-kalij ATP-aza; zebrica
• Keywords (english): cryosections; (immuno)fluorescence analysis; actin; sodium-potassium ATPase; zebrafish
• Language: croatian
• URN:NBN: urn:nbn:hr:217:014155
• Study programme: Title: Experimental Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra eksperimentalne biologije (magistar/magistra eksperimentalne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-09-29
• Created on: 2021-10-14 13:16:47