Naslov
Thick and cleared - Blood vessels and neurons can be visualized in the cleared mouse brain using inverted fluorescence microscopy

Autori
Hamer, Dominik ; Petrinec, Daniela ; Berecki, Monika ; Skukan, Laura ; Gajović, Srećko

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Book of Abstracts / Macan, Jelena ; Kovačević, Goran - , 2022, 32-33

Skup
4th Croatian Microscopy Congress

Mjesto i datum
Poreč, Hrvatska, 18-20.05.2022

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Domaća recenzija

Ključne riječi
blood vessels, neurons, clearing technique, mouse brain, flourescence microscopy

Sažetak
To visualize whole organs or whole body of humans or animals different imaging techniques can be applied. Novel light sheet fluorescence microscopy (LSFM) allows to visualize large samples, in particular whole organs of the laboratory animals. The variety of tissue clearing procedures are used for achieving sample transparency and the visualisation is based on the labelling of structures of interest by fluorescence. The main goal of this research was to image the structures in the cleared mouse brain with a special task to verify if the clearing procedure can be beneficial even if “classical” easily available fluorescent microscopes were used for sample visualisation. The inverted fluorescence microscope (The EVOS® FL Auto Imaging System, ThermoFisher Scientific) was used as a test instrument for this purpose. Previously naturally transparent parts of mouse embryos were imaged using fluorescence microscopy by our group.1, 2 To add a fluorescent marker it was applied to the brain of live mouse by stereotaxic injection. The fluorescent staining 10 % fluorescein solution (Fluorescite, Alcon) and Isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor 568 Conjugate (Invitrogen) was injected by help of stereotaxic apparatus (David KOPF Stereotaxic Instrument Small Animal Frame 5001 H7000). Moroever, mouse brains were isolated from two months old animals (Thy1-YFP-16 strain), which naturally expressed yellow fluorescent protein in neurons. As a third approach blood vessel visualization Lycopersicon Esculentum Lectin Texas Red (Invitrogen) was injected in the left heart ventricle of living mouse (Figure 1). In all three cases the mice were perfused by 1× PBS and 4 % formalin solution and subsequently cleared. For whole brain tissue clearing, three methods were used: ECi (optical clearing using ethyl-cinnamate), iDISCO (immunolabeling-enabled threedimensional imaging of solvent-cleared organs) and PEGASOS (polyethylene glycol- associated solvent system). Cleared mouse brain samples were cut on approximately 1 mm thick slices using mold (Alto Acrylic 1mm Mouse Brain Coronal 40-75gm, CellPoint Scientific), mounted subsequently on the glass slides in the drop of the final clearing solution, covered by coverslips, and imaged using inverted fluorescence microscope. The ECi method was preferred as the protocol for clearing lasted only one day and used chemicals were nontoxic. iDISCO clearing technique made brain slices brittle and difficult to handle. Even without using LSFM, it was possible to visualize fluorescently labelled structures in thick samples. It was still to be clarified if this type of imaging is suitable beside qualitative description of the samples, to quantitative measurements as well. In conclusion, the clearing of mouse brain produces thick slices suitable as well for imaging and analysis by fluorescence microscopy.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA