Immunohistochemistry Analysis of Mouse Tumors

Hemotaxylin & Eosin (H & E) staining of mouse organs and tumors was performed by the staff at the Anatomical Pathology Laboratory Services at The Royal Children's Hospital, Melbourne, Australia according to the standard H&E protocol [57 (link)]. Immunohistochemistry analysis of mouse tumors was performed on formalin fixed, paraffin embedded 4 μm sections of the xenografts and were stained using a Ventana Benchmark Immunostainer (Ventana Medical Systems, Inc, Arizona, USA) as described previously [3 (link), 20 (link)]. Negative controls were prepared by incubating each tumor section without primary antibodies. Sections of human breast tissue, high-grade ovarian tumors and human tonsils were used as positive controls to determine the staining efficacy of primary antibodies. Immunohistochemistry images were taken using an Aperio ImageScope (Leica Microsystems, Mt Waverly, Australia) with the associated digital pathology viewing software. DAB staining was measured using the open source image processing package Fiji (https://fiji.sc/) with a plug-in developed to recognize DAB staining for 10 randomly captured images per section. DAB staining intensity of the negative control was subtracted from the DAB staining of the antibody of interest to get a measure of DAB staining of interest.