Double Immunohistochemical Staining Method for HIF-1alpha and its Regulators PHD2 and PHD3 in Formalin-fixed Paraffin-embedded Tissues

摘要

Hypoxia-inducible factor (HIF-1alpha) is expressed in thenuclei of tumor cells under hypoxic conditions, and is regulated,in part, by cytoplasmic prolyl hydroxylases (PHDs). As HIF-1alphais selectively expressed in tumor cells, inhibitors are being devel-oped for cancer therapy. Although methods for the detection ofHIF-1alpha and PHDs are available, an immunohistochemical doublestaining method for these markers in individual tumor cells isnot available. For method development a human squamous cellcarcinoma (SCC) xenograft, A253, was used as a known positivecontrol tissue for HIF-1alpha in well-differentiated areas withoutmicrovessels. This laboratory showed that tumor cells in theseareas are strongly positive for hypoxia markers. Another human,poorly differentiated SCC xenograft, FaDu, without hypoxicareas, was used as a negative control. PHD2 and 3 immunostainingwas optimized individually using the human kidney. To optimizeHIF-1alpha detection the pressure cooker time for antigen retrieval,concentration of the primary antibody, amplification reagent, andDAB development time were decreased. Casein blocking furtherdecreased the background. Double staining resulted in brownnuclei for HIF-1alpha (DAB), and pink cytoplasmic staining forPHD2, 3 (fast red). The isotype-matched controls were negative.Normal human tissues had no detectable HIF-1alpha, but expressedPHD2, 3. The potential use of this new and improved method wasconfirmed by analyzing 15 surgical biopsies of oropharyngeal SCCof which 6 were positive for HIF-1alpha. This new method defined theoptimal conditions for detection of HIF-1alpha and PHDs in indivi-dual tumor cells and could have a diagnostic and therapeuticpotential.