Double immunohistochemical staining method for HIF-1alpha and its regulators PHD2 and PHD3 in formalin-fixed paraffin-embedded tissues.

Hypoxia-inducible factor (HIF-1alpha) is expressed in the nuclei of tumor cells under hypoxic conditions, and is regulated, in part, by cytoplasmic prolyl hydroxylases (PHDs). As HIF-1alpha is selectively expressed in tumor cells, inhibitors are being developed for cancer therapy. Although methods for the detection of HIF-1alpha and PHDs are available, an immunohistochemical double staining method for these markers in individual tumor cells is not available. For method development a human squamous cell carcinoma (SCC) xenograft, A253, was used as a known positive control tissue for HIF-1alpha in well-differentiated areas without microvessels. This laboratory showed that tumor cells in these areas are strongly positive for hypoxia markers. Another human, poorly differentiated SCC xenograft, FaDu, without hypoxic areas, was used as a negative control. PHD2 and 3 immunostaining was optimized individually using the human kidney. To optimize HIF-1alpha detection the pressure cooker time for antigen retrieval, concentration of the primary antibody, amplification reagent, and DAB development time were decreased. Casein blocking further decreased the background. Double staining resulted in brown nuclei for HIF-1alpha (DAB), and pink cytoplasmic staining for PHD2, 3 (fast red). The isotype-matched controls were negative. Normal human tissues had no detectable HIF-1alpha, but expressed PHD2, 3. The potential use of this new and improved method was confirmed by analyzing 15 surgical biopsies of oropharyngeal SCC of which 6 were positive for HIF-1alpha. This new method defined the optimal conditions for detection of HIF-1alpha and PHDs in individual tumor cells and could have a diagnostic and therapeutic potential.