Abstract
In these culture models, the normal cytoarchitecture and local neuronal circuits of the spinal cord are preserved, offering a compromise between dissociated cell cultures and complete animal studies. The addition of 5-ethynyl-2’-deoxyuridine (EdU) to the culture medium allows for the detection of proliferating cells.
Keywords: Neurogenesis, Spinal cord dissection, Ependymal cell, 5-ethynyl-2'-deoxyuridine
Materials and Reagents
Equipment
Procedure
Representative data
Following detection of EdU, these slices were also immunohistochemically labelled with anti-PKD2L-1 (polycystic kidney disease 2-like 1 protein) labelling cerebrospinal contacting neurones. Figure 8. An example taken of EdU labelling in the region surrounding the central canal of an organotypic cultured spinal cord slice
Notes
Minimising the time taken for the spinal cord to be removed from the animal and established in culture is imperative to achieving good viability in the slices. Therefore the preparation must be carried out as efficiently as possible, while still ensuring there is no damage to the spinal cord.
Recipes
Acknowledgments
This procedure known as the interface method was modified for spinal cord culture from the original method of Stoppini et al. (1991).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.
Sorry to hear you have had trouble with the slices not adhering to the membrane. The most likely reason for this is that all the media has not been removed after transferring the slices on to the membrane. Other occasion when I have trouble with the slices adhering to the filter is when the tissue is not viable, may be due to it taking a bit to long to complete the prep or bacterial contamination. I hope this is helpful