Multiplex Immunofluorescence Staining Protocol for the Dual Imaging of Hypoxia-Inducible Factors 1 and 2 on Formalin-Fixed Paraffin-Embedded Samples

Hypoxia is a common condition in rapidly proliferating tumors and occurs when oxygen delivery to the tissue is scarce. It is a prevalent feature in ~90% of solid tumors. The family of HIF (hypoxia-inducible factor) proteins—HIF1α and HIF2α—are the main transcription factors that regulate the response to hypoxia. These transcription factors regulate numerous downstream gene targets that promote the aggressiveness of tumors and therefore have been linked to worse prognosis in patients. This makes them a potential biomarker to be tested in the clinical setting to predict patient outcomes. However, HIFs have been notoriously challenging to immunolabel, in part due to their fast turnover under normal oxygen conditions. In this work, we developed a multiplexed immunofluorescence (mIF) staining protocol for the simultaneous detection of HIF1α and HIF2α in the same formalin-fixed paraffin-embedded (FFPE) tissue section.