Pimonidazole hydrochloride (Hypoxyprobe-1, Chemicon, and Temecula, CA) was administered at 60 mg/kg body weight in WT mice. This agent was dissolved in PBS and administered via the tail vein 1 hour before LC.17 (link) Lung and liver samples were collected 24 hours after LC and then frozen in optimal cutting temperature (OCT) mounting medium (Sakura Finetek, Torrance, CA). For detection of pimonidazole, sections were incubated with fluorescein isothiocyanate (FITC) conjugated to mouse IgG1 monoclonal antibody (FITC-Mab1). A 1:100 dilution of secondary rabbit anti-FITC horseradish peroxidase conjugated antibody in the Hypoxyprobe-1 Plus Kit was used (Hypoxyprobe, Inc, Burlington, MA).
Oct mounting medium
Manufactured by Sakura Finetek
Sourced in United States
The OCT mounting medium is a specialized solution used to prepare tissue samples for optical coherence tomography (OCT) analysis. It is designed to provide a stable and consistent environment for the tissue, enabling high-quality imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
1 plus kit, by Hypoxyprobe (1 mentions)
Rabbit anti calretinin, by Swant (1 mentions)
Mouse anti parvalbumin, by Swant (1 mentions)
Rabbit anti calbindin d28k, by Swant (1 mentions)
Goat anti mouse igg1, by Biotium (1 mentions)
Goat anti rabbit igg, by Biotium (1 mentions)
Goat anti doublecortin dcx, by Santa Cruz Biotechnology (1 mentions)
Donkey anti goat igg, by Biotium (1 mentions)
Axio imager m1, by Zeiss (1 mentions)
Rabbit anti ki67, by Abcam (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Shandon cryotome fse, by Thermo Fisher Scientific (1 mentions)
4 protocols using oct mounting medium
1
Detecting Tissue Hypoxia Using Pimonidazole
2
Immunohistochemical Analysis of Neural Markers
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Animals were deeply anesthetized with sodium pentobarbital, and were exsanguinated by intracardiac perfusion with phosphate buffered saline (PBS) followed by fixation with perfusion of 4% paraformaldehyde (PFA). Whole brain was post-fixed in 4% PFA for 1 h at 4°C and cryoprotected. Each sample embedded in O.C.T. mounting medium (Sakura Finetek) was cut into 12-μm-thick sections at around −20°C and stored on slide glasses at −80°C. Sections were incubated at 4°C over night with primary antibodies: rabbit anti-Ki-67 (1:500, Abcam), goat anti-doublecortin (DCX) (1:1,000, Santa Cruz), rabbit anti-calretinin (1:1,500, SWANT) rabbit anti-calbindin D-28 K (1:400, SWANT), and mouse anti-Parvalbumin (1:7,500, SWANT). Then, the sections were incubated at room temperature for 2 h with fluorescence-conjugated secondary antibodies (donkey anti-goat IgG (H + L, 1:1,000, Biotium), goat anti-rabbit IgG (H + L, 1:1,000, Biotium) or goat anti-mouse IgG1 (1:2,000, Biotium). Signals were detected with a fluorescence microscope (AxioImager M1, Zeiss).
3
Xenograft Tumor Hypoxia Assessment
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Animal protocols were approved by the Institutional Animal Care and Use Committee. Xenografts were formed by injecting 5×106 cells subcutaneously into the hind legs of 6–8-week old female nude mice (athymic nu/nu; NCI Frederick Cancer Research Institute, Frederick, MD, USA). When the xenografts reached ~10 mm in diameter, the mice were injected i.v. with pimonidazole (80 mg/kg; HPI, MA). Two hours later, Hoechst 33342 (25 mg/kg; Sigma-Aldrich) was i.v. injected, and 1 min later, the mice were sacrificed by CO2 breathing. Then, tumors were excised and snap-frozen in OCT mounting medium (Sakura Finetek USA Inc., Torrance, CA, USA). Frozen sections of 8-μm thickness were prepared for the immunohistochemical analysis.
4
Assessing HS-1793 Efficacy on Tumor Hypoxia and Perfusion
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For the single-injection efficacy of HS-1793 concerning the change in tumor hypoxia, pimonidazole (60 mg/kg) was administered for 4 h 15 min, HS-1793 (1.5 mg/kg) was administered for 2 h 15 min, and CCI-103F (60 mg/kg) was administered for 2 h before animal euthanasia (Fig. 2A). with regard to the single-injection efficacy of HS-1793 and the change in tumor perfusion, Hoechst 33342 (15 mg/kg) was administered for 26 min, HS-1793 (1.5 mg/kg) was administered for 16 min, and DiOC7 (1 mg/kg) was administered for 1 min before animal euthanasia (Fig. 2B). For the 30-day repeat injection efficacy of HS-1793 (0.5, 1 and 1.5 mg/kg), pimonidazole hydrochloride was injected intravenously (i.v.) into the tail vein at a dose of 60 mg/kg, and Hoechst 33342 was injected i.v. at a dose of 15 mg/kg in a total volume of 0.1 ml. Pimonidazole hydrochloride was administered for 2 h, and H33342 was administered for 1 min before animal euthanasia. After animal euthanasia, the tumor specimens were removed and frozen in OCT mounting medium (Sakura Finetek, Torrance, CA, USA). Next, consecutive 4-µm-thick frozen sections were cut on a Shandon Cryotome FSE (Thermo Fisher, waltham, MA, USA). The sections were then stored at -80˚C until staining.
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