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. 2019 Feb 5;9:1407. doi: 10.1038/s41598-018-38130-0

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© The Author(s) 2019

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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Construction and image analysis of a spheroid tissue microarray (microTMA). Fixed spheroids were embedded in a microTMA mold prior to paraffin embedding and sectioning on a microtome. TMA sections were then immunofluorescence stained and imaged using confocal scanning followed by dearraying of the spheroid images and image analysis (see material and methods section 4.8). (A) Schematic showing construction and processing of the microTMA for high-throughput histology and image analysis of 3D spheroid cultures. (B) Image analysis process to quantify tumor size and the number of cleaved caspase 3 or Ki67 positive tumor cells or normal neuronal cells. a dual immunofluorescence stained section showing GFP positive cells (red) and Ki67 positive cells (green), blue cells (DAPI) are normal (non-tumor cells); b - spectral unmixing and removal of autofluorescence; c, d - segmentation in the red channel to define tumor and normal cells/areas; e–h: segmentation in the green channel to define Ki67 or caspase positive cells, which are then counted in the tumor- and normal-cell regions.