Abstract
This microinjection protocol allows the manipulation and tracking of neural stem and progenitor cells in tissue at single-cell resolution. We demonstrate how to apply microinjection to organotypic brain slices obtained from mice and ferrets; however, our technique is not limited to mouse and ferret embryos, but provides a means of introducing a wide variety of membrane-impermeable molecules (e.g., nucleic acids, proteins, hydrophilic compounds) into neural stem and progenitor cells of any developing mammalian brain. Microinjection experiments are conducted by using a phase-contrast microscope equipped with epifluorescence, a transjector and a micromanipulator. The procedure normally takes ∼2 h for an experienced researcher, and the entire protocol, including tissue processing, can be performed within 1 week. Thus, microinjection is a unique and versatile method for changing and tracking the fate of a cell in organotypic slice culture.
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Acknowledgements
We thank J. Helppi and other members of the animal facility as well as H. Wolf of the workshop and K. Margitudis of the photo laboratory of the Max Planck Institute of Molecular Cell Biology and Genetics for excellent support; the staff of BioCrea, especially B. Langen, for ferret care and housing; M. Turrero-García for advice with ferret slice culture; F. Mora-Bermúdez for acquiring and processing the photographs of the microinjection equipment (Fig. 3a and Supplementary Fig. 1a,b); and Y.J. Chang, E. Lewitus and M. Wilsch-Bräuninger for helpful comments on the manuscript. W.B.H. was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (SFB 655, A2; TRR 83, Tp6) and the European Research Council (250197), by the DFG-funded Center for Regenerative Therapies Dresden, and by the Fonds der Chemischen Industrie.
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F.K.W. and E.T. designed and performed all microinjections and most other experimental work, analyzed the data and wrote the manuscript; C.H. performed experiments; E.T. and W.B.H. supervised the project and wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Microinjection pipette optimization.
Comparison between bad and good microinjection pipette. A good microinjection pipette has longer taper (a, b, arrows) and smaller tip (c, note the smaller angle), as compared to a bad one.
Supplementary Figure 2 Collagen embedding.
Comparison between optimal and suboptimal collagen embedding. Top row: photographs of glass-bottom Petri dishes containing organotypic slices embedded with too little, an optimal amount, or too much collagen. Bottom row: diagram illustrating optimal versus suboptimal collagen embedding (side view of Petri dish). Note the difference in the amount of collagen solution. All animal studies were conducted in accordance with the German animal welfare legislation.
Supplementary information
Supplementary Figure 1
Microinjection pipette optimization. (PDF 3869 kb)
Supplementary Figure 2
Collagen embedding. (PDF 38091 kb)
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Wong, F., Haffner, C., Huttner, W. et al. Microinjection of membrane-impermeable molecules into single neural stem cells in brain tissue. Nat Protoc 9, 1170–1182 (2014). https://doi.org/10.1038/nprot.2014.074
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DOI: https://doi.org/10.1038/nprot.2014.074
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