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. 2017 Jul 13;7:5297. doi: 10.1038/s41598-017-05573-w

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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The organotypic cerebellar slice–tumour co-culture. (A) Workflow for OCSC generation and tumour spheroid implantation. (1) Decapitation of mouse pup(s) at PND 8–10 and isolation of cerebellum. (2) Sectioning of cerebellum under physiological conditions using a vibratome to generate 350 µm thick sagittal or coronal sections. (3) Culture of cerebellum sections on membrane inserts for 15 days in vitro (DIV). (4) Implantation of one LifeAct enhanced GFP (LA-EGFP) expressing tumour spheroid per slice. (5) Further incubation of the co-culture in vitro without or with treatment. (6) Fixation and immunofluorescence for microscopy analysis. (7) Quantification and data analysis. (B) Sagittal and coronal sections of cerebella, stained with anti-Calbindin (red) to visualise Purkinje cells. (C) Implantation of LA-EGFP expressing DAOY cells spheroid on the slices. (D) Dissemination of tumour cells (green) in the cerebellar slices after 5 days post initiation of culture.