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University of Potsdam

6 ARTICLES PUBLISHED IN JoVE

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Biology

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development
Verónica A. Lombardo 1, Anje Sporbert 1, Salim Abdelilah-Seyfried 1
1Max Delbrück Center for Molecular Medicine

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

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Biology

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle
Cécile Otten 1, Salim Abdelilah-Seyfried 1
1Max Delbrück Center for Molecular Medicine

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

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Developmental Biology

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis
Verónica A. Lombardo *1,2,3, Cécile Otten *1,2, Salim Abdelilah-Seyfried 1,2,3
1Max Delbrück Center for Molecular Medicine, 2Institute of Biochemistry and Biology, University of Potsdam, 3Institute of Molecular Biology, Medizinische Hochschule Hannover

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

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Immunology and Infection

Generation of Murine Monoclonal Antibodies by Hybridoma Technology
Pamela Holzlöhner 1, Katja Hanack 1
1Department of Biochemistry and Biology, University of Potsdam

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

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Biochemistry

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts
Valentin Dunsing 1, Salvatore Chiantia 1
1Institute for Biochemistry and Biology, Cell Membrane Biophysics Group, University of Potsdam

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

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Chemistry

Single-Molecule Surface-Enhanced Raman Scattering Measurements Enabled by Plasmonic DNA Origami Nanoantennas
Amr Mostafa 1, Yuya Kanehira 1, Anushree Dutta 1, Sergio Kogikoski Jr. 1, Ilko Bald 1
1Institute of Chemistry, University of Potsdam

This protocol demonstrates single-molecule surface-enhanced Raman scattering (SERS) measurements using a DNA origami nanoantenna (DONA) combined with colocalized atomic force microscopy (AFM) and Raman measurements.

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