Journal article
Double Immunohistochemical Staining Method for HIF-1α and its Regulators PHD2 and PHD3 in Formalin Fixed Paraffin Embedded Tissues
Applied immunohistochemistry & molecular morphology, Vol.18(4), pp.375-381
07/01/2010
DOI: 10.1097/PAI.0b013e3181d6bd59
PMCID: PMC3215297
PMID: 20216402
Abstract
Hypoxia inducible factor (HIF-1α) expressed in the nuclei of tumor cells under hypoxic conditions, and is regulated, in part, by cytoplasmic prolyl hydroxylases (PHDs). Since HIF-1α is selectively expressed in tumor cells, inhibitors are under development for cancer therapy. Although methods for detection of HIF-1α and PHDs are available, an immunohistochemical (IHC) double staining method for these markers in individual tumor cells is not available. For the method development, human squamous cell carcinoma (SCC) xenograft A253 was used as a known positive control tissue for HIF-1α in well differentiated areas without microvessels. This laboratory demonstrated 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 human kidney. To optimize HIF-1α 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 background. The double staining resulted in brown nuclei for HIF-1α (DAB), and pink cytoplasmic staining for PHD2, 3 (fast red). The isotype matched controls were negative. Normal human tissues had no detectable HIF-1α, but expressed PHD2, 3. Potential utility of this new and improved method was confirmed by analyzing fifteen surgical biopsies of oropharyngeal SCC of which 6 were positive for HIF-1α. This new method defined optimal conditions for detection of HIF-1α and PHDs in individual tumor cells, and could have diagnostic and therapeutic potential.
Details
- Title: Subtitle
- Double Immunohistochemical Staining Method for HIF-1α and its Regulators PHD2 and PHD3 in Formalin Fixed Paraffin Embedded Tissues
- Creators
- Mary M. Vaughan - Roswell Park Cancer InstituteKaroly Toth - Roswell Park Cancer InstituteSreenivasulu Chintala - Roswell Park Cancer InstituteYoucef M. Rustum - Roswell Park Cancer Institute
- Resource Type
- Journal article
- Publication Details
- Applied immunohistochemistry & molecular morphology, Vol.18(4), pp.375-381
- DOI
- 10.1097/PAI.0b013e3181d6bd59
- PMID
- 20216402
- PMCID
- PMC3215297
- NLM abbreviation
- Appl Immunohistochem Mol Morphol
- ISSN
- 1541-2016
- eISSN
- 1533-4058
- Grant note
- R21 CA133682-01A2 || CA / National Cancer Institute : NCI
- Language
- English
- Date published
- 07/01/2010
- Academic Unit
- Hematology, Oncology, and Blood & Marrow Transplantation; Internal Medicine
- Record Identifier
- 9984359894602771
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