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10 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy
Ludovico Silvestri 1, Alessandro Bria 2,3, Irene Costantini 1, Leonardo Sacconi 1,4, Hanchuan Peng 5, Giulio Iannello 2, Francesco Saverio Pavone 1,4,6,7
1European Laboratory for Non-linear Spectroscopy (LENS), 2Integrated Research Centre, University Campus Bio-medico of Rome, 3DAEMI, University of Cassino, 4National Institute of Optics (CNR-INO), 5Allen Institute for Brain Science, 6Department of Physics, University of Florence, 7ICON Foundation, Sesto Fiorentino, Italy

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

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JoVE Core

A Simple Stimulatory Device for Evoking Point-like Tactile Stimuli: A Searchlight for LFP to Spike Transitions
Antonio G. Zippo 1, Sara Nencini 1, Gian Carlo Caramenti 2, Maurizio Valente 1, Riccardo Storchi 3, Gabriele E.M. Biella 1
1Institute of Molecular Bioimaging and Physiology (IBFM), Department of Biomedicine, National Research Council, 2Institute of Biomedical Technologies (ITB), Department of Biomedicine, National Research Council, 3Faculty of Life Sciences, University of Manchester

To elucidate the complex transition from Local Field Potentials (LFPs) to spikes a suitable stimulator for light mechanical peripheral stimuli was built. As an application, the spiking activities recorded from somatosensory cortex were analyzed by a multi-objective optimization strategy. The results demonstrated that the proposed stimulator was able to deliver tactile stimuli with millisecond and millimeter precisions.

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Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo
Anna L. Allegra Mascaro 1, Leonardo Sacconi 1,2, Francesco Saverio Pavone 1,2,3,4
1European Laboratory for Non-Linear Spectroscopy, University of Florence, 2National Institute of Optics, National Research Council, 3Department of Physics and Astronomy, University of Florence, 4International Center for Computational Neurophotonics (ICON Foundation)

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

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Biology

Silencing of BRCA2 to Identify Novel BRCA2-regulated Biological Functions in Cultured Human Cells
Loredana Moro 1, Nicoletta Guaragnella 1, Sergio Giannattasio 1
1Institute of Biomembranes and Bioenergetics, National Research Council

Gene silencing by siRNA represents a convenient experimental strategy to analyze BRCA2-dependent biological functions with immediate implications to better understand cancer biology. A method to efficiently silence BRCA2, along with the experimental procedure to detect and quantify changes in BRCA2 protein expression by immunoblotting in human cell lines, is presented.

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Medicine

Ultrasound-based Pulse Wave Velocity Evaluation in Mice
Nicole Di Lascio 1,2, Claudia Kusmic 2, Francesco Stea 2,3, Francesco Faita 2
1Institute of Life Science, Scuola Superiore Sant'Anna, 2Institute of Clinical Physiology, National Research Council, 3Department of Clinical and Experimental Medicine, University of Pisa

Arterial stiffness represents a key factor in cardiovascular disease and pulse wave velocity (PWV) can be considered as a surrogate index for arterial stiffness. This protocol describes an image processing algorithm for calculating PWV in mice based on ultrasound image processing that is applicable at different arterial sites.

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Cancer Research

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma
Marianna Vitiello 1,2, Claudia Kusmic 1, Francesco Faita 1, Laura Poliseno 1,2
1Institute of Clinical Physiology, National Research Council (IFC-CNR), 2Oncogenomics Unit, Core Research Laboratory, ISPRO

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

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Biology

Mesoscopic Optical Imaging of Whole Mouse Heart
Francesco Giardini *1, Erica Lazzeri *1, Camilla Olianti *1, Giada Beconi 1, Irene Costantini 1,2, Ludovico Silvestri 1,3,4, Elisabetta Cerbai 1,5, Francesco S. Pavone 1,3,4, Leonardo Sacconi 1,3,6
1European Laboratory for Non-Linear Spectroscopy, 2Department of Biology, University of Florence, 3National Institute of Optics, National Research Council, 4Department of Physics and Astronomy, University of Florence, 5Department of Neurosciences, Psychology, Drugs and Child Health, University of Florence, 6Institute for Experimental Cardiovascular Medicine, Faculty of Medicine, University of Freiburg

We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope.

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Photoalignment of Brilliant Yellow Films for Fully Tunable Liquid Crystal Pretilt
Colin McGinty 1, Jakub Kolacz 2, Christopher M. Spillmann 2
1Research Associateship Program, National Research Council, 2Naval Research Laboratory

The ability to pattern the liquid crystal (LC) pretilt angle at the LC-substrate interface with a single photoalignment material remains limited. The protocol here presents a method for accessing a large range of pretilt angles utilizing oblique exposures of brilliant yellow photoalignment films.

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Medicine

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals
Daniele Panetta 1, Maria Angela Guzzardi 1, Federica La Rosa 1, Federico Granziera 1,2, Domiziana Terlizzi 3, Claudia Kusmic 1, Patricia Iozzo 1
1CNR Institute of Clinical Physiology, 2Sant’Anna School of Advanced Studies, 3Fondazione Regione Toscana/CNR “G. Monasterio”

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

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Bioengineering

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing
Alessandro Migliara 1, Martino Alfredo Cappelluti 1, Francesca Giannese 2, Sara Valsoni 1, Alberto Coglot 1, Ivan Merelli 1,3, Davide Cittaro 2, Angelo Lombardo 1,4
1San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 2Center for Omics Sciences, IRCCS San Raffaele Institute, 3Institute for Biomedical Technologies, National Research Council, 4Vita-Salute San Raffaele University

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

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