UNLP 1 article published in JoVE Cancer Research Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures Felipe J. Núñez1,2, Flor M. Mendez2, Maria B. Garcia-Fabiani1,2, Joaquín Pardo1,3, Marta Edwards1,2, Pedro R. Lowenstein1,2, Maria G. Castro1,2 1Department of Neurosurgery, University of Michigan Medical School, 2Department of Cell & Developmental Biology, University of Michigan, 3INIBIOLP, Histology B-Pathology B, School of Medicine, UNLP Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.