Detection of Intratumoral Immune Cell Antigens in an Immunocompetent Glioblastoma Stem-like Cell Model Using Substrate-Based Immunohistochemistry​.

Glioblastoma (GBM) is the most common and lethal primary brain malignancy, lacking effective therapies. It is characterized by a profoundly immunosuppressive tumor microenvironment, driven in part by treatment-resistant GBM stem-like cells (GSCs). Here, we describe reproducible substrate-based single- and dual-color immunohistochemistry (IHC) protocols for detecting tumor-infiltrating immune cell-associated surface and intracellular antigens in formalin-fixed, paraffin-embedded (FFPE) brain tumor sections. The brain tumors were derived from mouse GSCs, which were orthotopically implanted in immunocompetent mice. The single-color IHC workflow uses a horseradish peroxidase (HRP)-based chromogenic detection system for visualization of individual immune cell antigens, whereas the dual-color IHC workflow employs a sequential alkaline phosphatase (AP)-based staining strategy for simultaneous detection of two immune cell antigens within the same tissue section, including workflows that use the same host origin primary antibodies. These methods provide a practical and reproducible framework for chromogenic analysis of tumor-infiltrating immune cell-associated antigens in GBM tissues and may be adaptable to other preclinical tumor models and potentially to human FFPE specimens.