Quantifying Tissue Hypoxia Levels

Tissue hypoxia levels were assessed by Hypoxyprobe immunofluorescence as described before54 (link)55 (link). Briefly, animals were administered Hypoxyprobe (Hypoxyprobe, Inc.) via intraperitoneal injection (60 mg kg⁻¹ body weight). Thirty minutes after injection, tissues were harvested, fixed overnight in 4% buffered formalin and embedded in paraffin. Tissue sections were deparaffined, rehydrogenated and incubated with anti-Hypoxyprobe (rabbit anti-PAb2627AP, 1:200 dilution Hypoxyprobe, Inc.) overnight at 4 °C. Hypoxia signalling was detected by applying Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1:1,000 dilution, Life Technologies). Quantification of the fluorescent signalling was performed using the Image-Pro Plus software (Media Cybernetics, Bethesda, MD). The density of the fluorescence was measured. The average densities of 20 areas per samples were determined and the s.e.m. is indicated.

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Sun K., Zhang Y., D'Alessandro A., Nemkov T., Song A., Wu H., Liu H., Adebiyi M., Huang A., Wen Y.E., Bogdanov M.V., Vila A., O'Brien J., Kellems R.E., Dowhan W., Subudhi A.W., Jameson-Van Houten S., Julian C.G., Lovering A.T., Safo M., Hansen K.C., Roach R.C, & Xia Y. (2016). Sphingosine-1-phosphate promotes erythrocyte glycolysis and oxygen release for adaptation to high-altitude hypoxia. Nature Communications, 7, 12086.

Publication 2016

Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody Image-Pro Plus software

Alexa fluor 488 Animals Anti igg Antibody Body weight Dilution Donkey Embedded paraffin Fluorescence Formalin Hypoxia Immunofluorescence Intraperitoneal injection Rabbit Tissue

Corresponding Organization : Central South University

Other organizations : The University of Texas Health Science Center at Houston, University of Colorado Denver, University of Oregon, Virginia Commonwealth University

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Variable analysis

independent variables

Hypoxyprobe (Hypoxyprobe, Inc.) administration via intraperitoneal injection (60 mg kg^-1 body weight)

dependent variables

Tissue hypoxia levels assessed by Hypoxyprobe immunofluorescence

control variables

Thirty minutes after Hypoxyprobe injection, tissues were harvested, fixed overnight in 4% buffered formalin and embedded in paraffin
Tissue sections were deparaffined, rehydrogenated and incubated with anti-Hypoxyprobe (rabbit anti-PAb2627AP, 1:200 dilution Hypoxyprobe, Inc.) overnight at 4 °C
Hypoxia signalling was detected by applying Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1:1,000 dilution, Life Technologies)
Quantification of the fluorescent signalling was performed using the Image-Pro Plus software (Media Cybernetics, Bethesda, MD)
The density of the fluorescence was measured, and the average densities of 20 areas per samples were determined with the s.e.m. indicated

controls

No positive or negative controls were explicitly mentioned in the protocol.

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