Immunohistochemical Analysis of Tumor Samples

Tumor samples excised from the mice, washed with PBS, placed in 10% buffered formalin for 24 h, and transferred to 70% ethanol for histopathological analysis. Immunohistochemistry was performed by Histowiz Inc. (histowiz.com) using a Standard Operating Procedure and fully automated workflow. Samples were processed, embedded in paraffin, and sectioned at 4μm [16 (link)]. Immunohistochemistry was performed on a Bond Rx autostainer (Leica Biosystems) with enzyme treatment (1:1000) using standard protocols. Antibodies used were rat monoclonal F4/80 primary antibody (eBioscience, 14–4801, and 1:200) and rabbit anti-rat secondary (Vector, 1:100) [17 (link)–19 (link)]. Bond Polymer Refine Detection (Leica Biosystems) was used according to the manufacturer’s protocol. After staining, sections were dehydrated, and film cover slipped using a Tissue-Tek Prisma and Cover-slipper (Sakura). Whole slide scanning (40×) was performed on an Aperio AT2 (Leica Biosystems).