Immunohistochemical staining with CD34 or pimonidazole was performed on formalin-fixed and paraffin-embedded tissue sections. Briefly, after antigen retrieval, tissue sections were incubated with anti-CD34 antibody (Abcam, USA) overnight at 4°C, and then incubated with biotinylated secondary antibody, and then with avidin–biotin–peroxidase complex (DAKO, Glostrup, Denmark). Finally, the tissue sections were incubated with 3′, 3′-diaminobenzidine (Sigma, St Louis, MO, USA) and counterstained with hematoxylin. In negative controls, primary antibodies were not applied. Pimonidazole hydrochloride (Chemicon International, Temecula, CA, USA) was used to detect the tumor hypoxia as previously described.23 (link) Briefly, tumor-bearing mice were intraperitoneally injected with Pimonidazole hydrochloride (0.1 mg/g body weight) dissolved in 10 mg/ml in 0.9% saline 1 h before sacrifice. The ratio of pimonidazole-positive area (%) was defined as the pimonidazole-positive area divided by the visible tumor area under 100-fold magnifications. The tumor micro-vessel density (MVD) was expressed as the ratio of CD34 positive stained area per total tumor area in a 200× high-power field.
Pimonidazole hydrochloride
Manufactured by Merck Group
Sourced in United States
Pimonidazole hydrochloride is a hypoxia probe used in research applications. It is a chemical compound that selectively binds to cells in hypoxic (low oxygen) environments, allowing for the identification and quantification of hypoxic regions within biological samples.
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3 protocols using pimonidazole hydrochloride
1
Tumor Hypoxia and Angiogenesis Assessment
2
Hypoxia Quantification in Xenograft Tumors
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Mice bearing HCCLM3 xenografts were injected intravenously with pimonidazole hydrochloride at 60 mg/kg (Chemicon International, Temecula, CA) 90 minutes before euthanasia. The xenografts were then removed and immediately fixed in 10% buffered formalin in the dark. Slides (4mm thick) with peritumoral tissue preparations were incubated with polyclonal rabbit antiserum to pimonidazole-protein adducts, which were visualized with 3, 3-diaminobenzidine. Area fractions showing pimonidazole staining were determined by image analysis.
3
Peritoneal Hypoxia and GCSPC Distribution
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Eight- to ten-week-old male BALB/c nude mice (weighing 18–20 g each) were obtained from Vital River (Beijing, China). All animal protocols received prior approval from the China Medical University Animal Ethics Committee, and all experiments were performed in accordance with relevant guidelines and regulations.
To investigate the peritoneal hypoxic region and GCSPC distribution, a group of 15 mice were intraperitoneally (i.p.) injected with 1 × 104 GC1sps to establish a time-dependent GCPD model. Peritoneal tumor-bearing mice were sacrificed on days 7, 14, and 21 (five mice at each time point). The peritoneal hypoxic region was assessed by pimonidazole incorporation, as described previously 29 (link). Briefly, 60 mg/kg pimonidazole hydrochloride (Chemicon) was injected intravenously (i.v.) into tumor-bearing mice. Two hours after injection, mice were sacrificed by cervical dislocation, and the peritoneal greater omentum was fixed and sectioned. The peritoneal hypoxic region was assayed by immunofluorescence confocal staining with a rabbit polyclonal anti-pimonidazole antibody (Chemicon).
To investigate the peritoneal hypoxic region and GCSPC distribution, a group of 15 mice were intraperitoneally (i.p.) injected with 1 × 104 GC1sps to establish a time-dependent GCPD model. Peritoneal tumor-bearing mice were sacrificed on days 7, 14, and 21 (five mice at each time point). The peritoneal hypoxic region was assessed by pimonidazole incorporation, as described previously 29 (link). Briefly, 60 mg/kg pimonidazole hydrochloride (Chemicon) was injected intravenously (i.v.) into tumor-bearing mice. Two hours after injection, mice were sacrificed by cervical dislocation, and the peritoneal greater omentum was fixed and sectioned. The peritoneal hypoxic region was assayed by immunofluorescence confocal staining with a rabbit polyclonal anti-pimonidazole antibody (Chemicon).
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