Abstract
Transplantation in mouse brain slices is a powerful tool in order to study axon targeting and migrational events during development. Taking advantage of donors and recipients belonging to different genotypes, this technique allows researchers to assess the contribution of donor and/or recipient tissue by performing various combinations and to study cell-autonomous functions or effects that are influenced by the recipient’s environment (Bastakis, et al., 2015). Here we describe the transplantation procedure on sagittal brain slices containing olfactory bulb (OB). Specifically, we have transplanted the proximal-to-the-cortex part of dorsal OB to the same region on a recipient slice. Transplanted slices can be cultured for up to 3 days before their morphology is disfigured due to growth in 3D. Re-sectioning of these slices allows for a more detailed immunohistochemical analysis.
Materials and Reagents
Equipment
Note: All surgical tools should be UV-sterilized before use.
Procedure
Housing and all animal procedures used were according to the European Union policy (Directive 86/609/EEC) and institutionally approved protocols. E0.5: day of the vaginal plug.
Representative data
Representative images of the expected results regarding migration of projection neurons (Tbr1+) from the proximal-to-the-cortex part of dorsolateral OB derived from a GFP-labelled mouse (BrdU pulsed at E11.5) is shown below (Figure 4, adapted from Bastakis et al., 2015). Figure 4. Transplantation of dorsal OB from mouse brain sections for the study of migrating projection neurons. A. Transplants were generated on sagittal E14.5 sections as shown in the scheme, with donor cells expressing GFP or being BrdU-pulsed at E11.5. B. WT GFP+, BrdU+ (BrdU injection at E11.5) cells were transplanted on WT recipients and immunostained for GFP and Tbr1 (marker of immature OB projection neurons) or GFP and BrdU. Migrating cells (GFP+) were projection neurons (co-localization with Tbr1, arrowheads, upper images) and were born at E11.5 (co-localization with BrdU, arrowheads, lower images). The dorsal part of the slice is shown in B. Scale: 150 μm.
Recipes
Acknowledgments
This protocol was adapted from the previously published study, de Diego et al. (2002) and it was performed by Bastakis et al. (2015). This work was supported by the European Commission FP7 programme “Translational Potential” [contract number 285948], InnovCrete [316223], ARISTEIA I [Project 593 MyelinTag] and by Institute of Molecular Biology and Biotechnology intramural grants.
References
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