Kit to Demonstrate Hypoxia In Vivo

September 11, 2018

The Scripps Research Institute
The Cell and Molecular Biology
Assistant Professor

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Company:

Hydroxyprobe

Product Name:

Hypoxyprobe-Red549 Kit

Catalog Number:

HP7-100Kit

High levels of hypoxia in vivo are associated with the induction of tumor angiogenesis, which in turn, is crucial for primary tumor growth and cancer cell dissemination to the secondary sites. To verify whether angiogenic vessels that sustain tumor cell dissemination are formed in response to high levels of hypoxic factors, we employed Hypoxyprobe-Red549 Kit in vivo in the chick embryo human xenograft model. We did not detect any hypoxia areas in the developing primary tumors, but hypoxic areas were detected in the embryos treated with cobalt chloride as a hypoxia mimetic agent.

Experimental Design and Results Summary

Application

Immunohistochemstry after in vivo treatment with a hypoxia-sensitive probe

Starting Material

Chicken embryos with the human primary tumors

Protocol Overview

Chicken embryos were incubated ex ovo and grafted with the GFP-tagged human tumor cells. After 5-7 days, a bioreactive chemical probe, pimonidazole (a part of the kit) was inoculated i.v. at 7.5 mg/0.2 mL PBS per embryo. For positive control, the developing tumors were treated topically with CoCl2 solution before piminidazole injections. After 2-hr incubation period that is required for the formation of pimonidazole adducts in hypoxic cells, the tissue containing human tumors was fixed in Zn-buffered formalin. After overnight fixation, tissue samples were washed in PBS, permeabilized in 1% Triton X-100 for 2 hr at RT, and blocked in 10% goat serum and 5% BSA in 0.1% Triton X-100. Tissue samples were stained overnight at 4oC with the DyLight 549-conjugated mouse anti-piminodazole antibody 4.3.11.3. (part of the kit) at 20 microgram per mL in PBS supplemented with 0.1% BSA and 0.1% Triton X-100. After washing in PBS, tissue samples were evaluated using immunofluorescence microscopy.

Tips

The probe might be not sensitive enough in vivo, especially if the levels of hypoxia are not high enough or the probe is not efficiently delivered to hypoxic areas. The use of cobalt chloride solution may provide a reliable positive control.

Results Summary

In tumors, hypoxia was detected as a yellow signal after merging monochromatic images acquired for GFP-tagged tumor cells (green channel) and pimonidazole adducts (red channel).

DOI or PMID #

N/A

Additional Notes

We were able to detect hypoxia in vivo only after treatment of tumors with a hypoxia-inducing agent.

Image Gallery

Summary

The Good

After multiple adjustments of treatment and staining protocols, hypoxic areas could be detected in vivo.

The Bad

We were able to detect hypoxia only after treatment of tissues with a strong hypoxia-indicing agent.

The Bottom Line

The the kit could be used for detection of hypoxia in vivo, but it might require some level of determination for finding right combinations of reagent concentrations and incubation time.

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