To evaluate the tumor hypoxia at the early stage in PANC1-bearing BALB/c nude mice, immunofluorescence analysis was conducted using a HypoxyprobeTM−1 Omni Kit (HP3−100; Hypoxyprobe, Burlington, MA, USA). Pimonidazole HCl (Hypoxuprobe-1, 60 mg/kg) was injected intraperitoneally 1 h before they were killed. Staining was performed according to the manufacturer’s instructions. The tissue sections were cut and fixed in cold acetone for 10 min. The sections were rinsed and incubated overnight at 4 °C with rabbit anti-pimonidazole antibodies (PAb2627AP, 1:100). The sections were then incubated for 2 h with FITC-conjugated goat-anti-rabbit antibody (Thermo, 1:1000). Between all steps of the staining procedure, the sections were rinsed three times with for 5 min in PBS and imaged by CLSM.
1 omni kit
Manufactured by Hypoxyprobe
Sourced in United States
The Hypoxyprobe-1 Omni Kit is a laboratory tool used to detect and measure hypoxia, or low oxygen levels, in biological samples. The kit contains reagents and accessories necessary for the immunohistochemical detection of hypoxic cells using the Hypoxyprobe-1 (pimonidazole) marker.
Automatically generated - may contain errors
Lab products found in correlation
Fv10i doc confocal laserscanning microscope, by Olympus (2 mentions)
Hematoxylin, by Roche (2 mentions)
Bluing reagent, by Roche (2 mentions)
Tissue tek oct compound, by Sakura Finetek (2 mentions)
Bz 9000 microscope, by Keyence (2 mentions)
Pan keratin clone c11, by Thermo Fisher Scientific (2 mentions)
Image pro software, by Media Cybernetics (2 mentions)
Nb100 417, by Novus Biologicals (1 mentions)
Pimonidazole, by Hypoxyprobe (1 mentions)
Dylight 594, by Jackson ImmunoResearch (1 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Protein block serum free reagent, by Agilent Technologies (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Anti γh2ax antibody, by Abcam (1 mentions)
Fv1200 confocal microscope, by Olympus (1 mentions)
Diaminobenzidine, by Merck Group (1 mentions)
Fluorophore labeled secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Ultrasensitive s p kit, by Maixin Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
17 protocols using 1 omni kit
1
Evaluating Tumor Hypoxia in PANC1 Mouse Model
2
Detecting Hypoxic Areas in Cutaneous I/R Injury
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Hypoxic areas after cutaneous I/R injury in I/R site were detected using the HypoxyprobeTM-1 Omni kit (Hypoxyprobe, Inc., Burlington, MA) according to the manufacture’s protocol, and as described previously9 (link). Pimonidazole HCl was injected intraperitoneally (60 mg/kg) 30 minutes before the sacrifice of the mice. Murine skins were removed and 4μm frozen sections were prepared and fixed in cold acetone (4 °C) for 10 minutes. Sections were incubated overnight at 4 °C with rabbit anti-pimonidazole Ab (PAb2627) diluted 1:20 in PBS containing 0.1% bovine serum albumin and 0.1% Tween 20. Sections were incubated for 1 hour with Alexa 488-conjugated secondary Ab. Images (8 fields/section) were taken and visualized with a FV10i-DOC confocal laserscanning microscope (Olympus). The positive area was determined by Image J (version1.48, NIH, Bethesda, MD) in the field (x600) as previously reported8 (link).
3
In Vivo Tumor Hypoxia Imaging
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A cocktail of either AF546-pHLIP + IR800-pHLIP (5 nmol each) or that of IR800-pHLIP + Cy5.5-RaftRGD (5 and 10 nmol, respectively) was administered to the mice and the mice were examined at 4 h post-injection. Pimonidazole, a commonly used exogenous hypoxia marker in histochemistry (1.5 mg, Hypoxyprobe-1 Omni Kit; Hypoxyprobe, Burlington, MA, USA), was administered intravenously to the mice 1 h before euthanizing. Tumors were removed and cryo-sectioned in sequential sections (10 µm thickness) as previously described [40 (link)]. Sections were stored in the dark at −80 °C until subsequent experiments.
4
Hypoxia Immunohistochemistry Protocol
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The hydroxyprobe reaction was carried out according to the manufacturer's protocol. Briefly, Hypoxyprobe-1 Omni kit (Hypoxyprobe, Inc. PAb2627AP, 1/200) was applied on every sample at room temperature for 60 min. Sections were then incubated with a specific secondary anti-rabbit biotin conjugated for 32 min at room temperature. Finally, sections were counterstained with Hematoxylin and Bluing reagent for 4 min each (Ventana Medical Systems, Roche, Tucson, AZ).
5
Hypoxia Immunohistochemistry Protocol
6
Histological Analysis of Hypoxic Brain Tissue
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Excised rat brains were fixed in neutral-buffered formalin (4%) before embedding in paraffin wax. Sections (7 µm) were cut, mounted onto poly-L-lysine-coated slides, and heat-fixed for 30 min at 58 °C. Paraffin was removed and sections were rehydrated using sequential immersions in 100% xylene, 100% ethanol, 80% ethanol, 50% ethanol and 100% distilled water. At this point, hematoxylin and eosin (H&E) staining was performed. Following an additional antigen retrieval step, staining for hypoxia was performed using pimonidazole in accordance with the manufacturer’s instructions (Hypoxyprobe™-1 Omni Kit, Hypoxyprobe, Burlington, MA, USA). All images were acquired on a M8 Microscope and Scanner (Precipoint, Freising, Germany).
7
Detecting Tumour Hypoxia via Pimonidazole
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Tumour tissue hypoxia was detected by Hypoxyprobe-1 Omni Kit (Hypoxyprobe Inc.), according to the manufacturer’s protocol. Mice were i.p. injected with 60~120 mg kg−1 pimonidazole HCl (25 mg ml−1 in 0.9% NaCl). At 60 min after injection, tumours were collected and fixed in 4% PFA overnight, followed by washing in PBS, and then equilibrated in 30% sucrose and embedded in OCT for frozen sectioning. Hypoxyprobe was visualized by rabbit anti-pimonidazole antisera and Alexa donkey anti-rabbit antibody.
8
Quantifying Hypoxia and Angiogenesis in Tumor Tissues
9
Detecting Tissue Hypoxia with Hypoxyprobe-1 Kit
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The Hypoxyprobe-1 Omni kit (Hypoxyprobe Inc., Burlington, MA) was used for immunochemical detection of tissue hypoxia. Briefly, mice were injected intraperitoneally with 60 mg/kg pimonidazole HCl 30 min prior to euthanasia. Following euthanasia, infected tissues were fixed in 10% formalin, paraffin embedded, and sectioned (5 µm). Unstained sections were deparaffinized with a series of xylene and ethanol washes, followed by antigen retrieval in boiling 10 mM sodium citrate buffer (pH 6). Tissues were blocked with 10% donkey serum (Jackson ImmunoResearch, West Grove, PA) and subsequently incubated with anti-Hypoxyprobe PAb2627AP (Hypoxyprobe Inc.). The primary antibody was detected by incubation with a biotinylated donkey anti-rabbit antibody, followed by incubation with streptavidin-conjugated Dylight 594 (Jackson ImmunoResearch). Tissues were mounted with ProLong antifade gold containing 4′,6-diamidino-2-phenylindole (Invitrogen, Grand Island, NY) and imaged on an Olympus BX60 fluorescence microscope with iVision software v.4.0.0 (BioVision Technologies, New Minas, Nova Scotia, Canada).
10
Quantifying Hypoxia and DNA Damage in Tumor Tissues
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Tumor tissues were excised 1 hour after intravenous injection of pimonidazole (60 mg/kg, Hypoxyprobe Inc.), frozen with Tissue-Tek O.C.T. compound (Sakura Finetek USA Inc.) by ultra-cold ethanol and sectioned (10 mm). After fixing with 4 % paraformaldehyde, sections were treated with cold acetone for 15 minutes. After blocking nonspecific-binding sites on sections with Protein Block Serum-Free reagent (Dako North America Inc.) for 30 minutes, the slides were covered by pimonidazole antibody (Hypoxyprobe-1 Omni Kit, Hypoxyprobe Inc.; 1:250) overnight at 4 C. The sections were then incubated with Alexa Fluor 488 secondary antibody for 1 h at room temperature, before being mounted with Prolong Gold antifade reagent with DAPI (Invitrogen). For γH2AX staining, anti-γH2AX antibody (Abcam, Inc.; 1:250) was used, followed by same staining procedures as above. The stained slides were scanned using a BZ-9000 microscope (Keyence), and the immunostain-positive area was quantified using ImageJ software (downloaded from https://imagej.nih.gov/ij/ ).
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