Hypoxia-Specific Tumor Labeling Protocol

For bioreductive labelling of hypoxic tissue areas, tumor bearing mice were i.v. injected with 60 mg/kg bodyweight pimonidazole (Hypoxyprobe™-1), diluted in isotonic saline. After 30 min, animals were perfused. To counterstain intracellular pimonidazole-adducts, wholemount staining of 1 mm vibratome sections or an intact BVZ-treated tumor xenograft was performed via an ATTO 594-labelled rat monoclonal α-pimonidazole antibody (clone 11.23.22.R; Hypoxyprobe Inc., Burlington, USA). Optically cleared, stained tissue samples were visualized by LSFM.

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Bauer N., Beckmann D., Reinhardt D., Frost N., Bobe S., Erapaneedi R., Risse B, & Kiefer F. (2024). Therapy-induced modulation of tumor vasculature and oxygenation in a murine glioblastoma model quantified by deep learning-based feature extraction. Scientific Reports, 14, 2034.

Publication 2024

Corresponding Organization :

Other organizations : University of Münster, Max Planck Institute for Molecular Biomedicine

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Variable analysis

independent variables

Dose of pimonidazole (Hypoxyprobe™-1) administered intravenously (60 mg/kg bodyweight)

dependent variables

Pimonidazole-adduct formation and localization in hypoxic tissue areas
Visualization of pimonidazole-adducts by LSFM

control variables

Time between pimonidazole injection and animal perfusion (30 minutes)
Use of isotonic saline as the vehicle for pimonidazole dilution
Counterstaining of pimonidazole-adducts using ATTO 594-labelled rat monoclonal α-pimonidazole antibody
Tissue sample preparation (1 mm vibratome sections or intact BVZ-treated tumor xenograft)

positive control

Not explicitly mentioned

negative control

Not explicitly mentioned

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