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Hypoxia Detection Methods

Methods to monitor hypoxia often rely on strategies for the detection of HIF protein expression. HIFs play central roles in the response to low-oxygenation, becoming stabilized and more prevalent under hypoxia. Monitoring HIF-alpha levels and prevalence within cells and tissues provides a measure of the extent of hypoxia and hypoxic gradients. Additionally, hypoxia is monitored by analyzing the expression of key proteins regulated by HIFs (e.g., CA IX, VEGF). This approach is particularly effective for proteins with longer half-lives than HIF proteins.


hypoxia proteins hypoxia CA IX VEGF Culture Models

Monitoring HIF- alpha Proteins

Under normoxic conditions, HIF-alpha subunits are rapidly targeted for degradation by the proteasomal system. Under hypoxia, HIF-alpha subunits are stable resulting in increased protein levels, particularly in the nucleus. View results of this increase in HIF-alpha subunits in the immunoassays, WB and ICC, below.

Explore top cited HIF-1 alpha antibody NB100-105: over 700 citations

HIF-1 and HIF-2 alpha Protein Levels Under Normoxia and Hypoxia  WB

Western Blot: HIF-2 alpha/EPAS1 Antibody [NB100-122] - Analysis of HIF-2 alpha in MDA-MB-231 cell lysate (overexpression and endogenous samples) using anti-HIF-2 alpha antibody. The data shows that HIF-2 alpha antibody does not cross-react with HIF-1 alpha overexpression. Additionally, elevated levels of HIF-1 and HIF-2 alpha were detected following incubation of MDA-MB-231 cells under hypoxic conditions.



HIF-1 alpha Protein Stabilization by Cobalt ICC

Immunocytochemistry/Immunofluorescence: HIF-1 alpha Antibody [NB100-449] - Formaldehyde-fixed asynchronous HeLa cells.

View Western Blot Protocol Specific for HIF-1alpha Antibody (NB100-105)

View Protocol: Hypoxic and Simulated-Hypoxic Cell Lysate Preparations


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Monitoring CA IX

Carbonic anhydrase IX (CA IX) is a transmembrane glycoprotein induced under hypoxic conditions. Induction of CA IX expression is regulated by HIFs via interaction with an HRE sequence within the 5’ promoter region of CA IX gene leading to increased transactivation.

Ca IX Protein Expression in Human Glioma under Normoxia and Hypoxia ICC

Carbonic anhydrase IX serves as a marker of hypoxia. Immunocytochemistry/Immunofluorescence: Carbonic Anhydrase IX/CA9 Antibody [NB100-417] - Analysis using the DyLight 488 conjugate of NB100-417. Staining of membrane cytoplasm Carbonic Anhydrase IX (red) in human glioma U87 cells. DAPI counterstains nuclei (blue).


CA IX is frequently expressed in tumor cells (e.g., clear cell renal carcinoma) and its expression is associated with poor prognosis. Because CA IX catalyzes the metabolism of CO2 to carbonic acid its induction under hypoxic conditions is associated with tumor acidification. Induction of CA IX expression is regulated by HIFs via interaction with an HRE sequence within the 5’ promoter region of CA IX gene leading to increased transactivation.


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Monitoring VEGF

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, the process by which new vessels are formed from pre-existent vasculature. Induction of angiogenesis under hypoxic conditions is common in tumors and represents an adaptation for cancer cell survival. VEGF and its receptors VEGF-R1, VEGF-R2 and VEGF-R3 are induced under hypoxia and play critical roles in the formation of new tumor vasculature. Monitoring VEGF/VEFG-R expression serves as a marker for hypoxia.

VEGF Expression in Human Angiosarcoma IHC

Immunohistochemistry-Paraffin: VEGF Antibody (VG1) [NB100-664] - IHC analysis of a formalin-fixed paraffin embedded human angiosarcoma tissue section using VEGF antibody (clone VG1). The endothelial cells of the blood vessels and most of the cancer cells showed strong positivity for VEGF protein.


VEGF R2/KDR/Flk‑1 Protein Expression in Human Kidney IHC

VEGF R2/KDR/Flk‑1 was detected in immersion fixed paraffin-embedded sections of human kidney using Goat Anti-Human VEGF R2/KDR/Flk‑1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF357) at 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Lower panel shows a lack of labeling if primary antibodies are omitted and tissue is stained only with secondary antibody followed by incubation with detection reagents. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.


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Hypoxia in 2D and 3D Culture Models

Hypoxia is common in tumors and occurs as the result of increased cell proliferation and metabolism, as well as deficiencies in blood supply and reduced tissue-oxygen diffusion. These factors continually expose tumor cells to fluctuating oxygenation which is difficult to replicate in the laboratory setting.

Tumor Oxygen Gradient and HIF-alpha Stability

View Hypoxic and Simulated-Hypoxic Cell Lysate Protocol

2D cell culture model systems have been predominantly used to study hypoxic responses. Most studies rely on culturing homogeneous cancer cell lines on flat surfaces that do not replicate the biologically significant extracellular environment.

Additional limitations of monolayer cell cultures include:

  • Cell-cell interactions are unlike those found in vivo
  • Cells acquire abnormal morphology
  • Cells have atypical distribution of membrane proteins

Various studies have shown that signaling pathways activated and resulting cellular adaptations that occur under hypoxia are influenced by the way cells are cultured in vitro. Therefore, 3D spheroid cell cultures have progressively become more accepted as model systems that more faithfully replicate the tumor microenvironment.

HIFs are stabilized in spheroids according to graded oxygen levels, similar to hypoxic tumor regions, reaching maximal expression in cells peripheral to the core. Several types of 3D spherical cultures have been developed that are reviewed in the white paper, A Technical Perspective: Understanding the Cellular Response to Hypoxia through In Vitro Model Systems


Modeling Hypoxia via Multicellular Tumor Spheroids


Explore Spheroid Models for Cancer and Hypoxia        Learn more about 3D Culture Models


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