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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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Morphological assessments of the cerebellar slices and co-culture in vitro. (A) Changes in the Purkinje cell monolayer upon in vitro culturing of the slices was monitored at DIV 0, DIV 1, DIV 10 and DIV 21 with anti-calbindin antibody (red) using confocal microscopy. (B) Grey scale images of cerebellar slices at DIV 0 and after being cultured for DIV 21. (C) Timeline of the experimental setup for five-day co-culture experiment. DIV 15 was selected as the time point suitable for the implantation of tumour spheroid. (D) The cerebellar cortex layers visualised using confocal microscopy at DIV 0 and DIV 15 for comparison with anti-Calbindin (red, Purkinje cell layer) and anti-NeuN (green, granular layer) antibodies. (E) Visualisation of the vasculature using anti-collagen IV (grey) at DIV 0 and DIV 15. (F) Determination of apoptosis and proliferation after five-day co-culture using anti-cleaved caspase 3 (red) and Click-iT EdU (blue), respectively. (G) Monitoring the tumour cells infiltrating the cerebellar slices. (H) Visualization of the vasculature (blue) in the presence of tumour cells. Lower panels: zoomed versions of boxed insets in F, G and H respectively. In H, EGFP and Collagen IV fluorescence was volumised.