HP4-100Kit: Hypoxyprobe-F6 Kit (100 mg CCI-103F (F6) plus 1 unit of anti-F6 rabbit antisera)
 
HP4-200Kit: Hypoxyprobe-F6 Kit (200 mg CCI-103F (F6) plus 1 unit of anti-F6 rabbit antisera)
 
HP4-1000Kit: Hypoxyprobe-F6 Kit (1000 mg CCI-103F (F6) plus 2 units of anti-F6 rabbit antisera)
 
General information:
 
Hypoxyprobe™ Kits have been used in over 350 published studies. Applications include studies of hypoxia in normal tissue physiology, pathophysiology and malignancy.
Hypoxyprobe™ Kits contain a choice of mouse monoclonal, FITC-conjugated mouse monoclonal or rabbit antibodies and can be studied by means of immunofluorescence, immunoperoxidase, immunoblotting, western analysis, flow cytometry or ELISA of isolated cells in vitro or tissues in vivo.
 
Hypoxyprobe™ Kits
Hypoxyprobe™ kits consist of two parts.
Hypoxyprobe™-1 is a substituted 2-nitroimidazole whose chemical name and only ingredient is 1-[(2-hydroxy-3-piperdinyl)propyl]-2-nitroimidazole hydrochloride. The free base of Hypoxyprobe™-1 is also known as Ro 03-8799 or pimonidazole.
Hypoxyprobe™-1 has a molecular weight of 290.7; a water solubility of 116 mg/mL which is equivalent to 400 millimolar and an ultraviolet absorbance maximum at 324 nm with an extinction coefficient of 7400 (free base) in 0.9% saline. The free base, pimonidazole, has a molecular weight of 254.3, a pKa of 8.7 and an octanol water partition coefficient of 8.5. At the concentration of 34 millimolar in 0.9% saline that is used for clinical application, Hypoxyprobe™-1 solutions have a pH of 3.9 ± 0.1. Hypoxyprobe™-1 has great chemical stability in solid form and in aqueous solutions and requires no stabilizer. For example, solid Hypoxyprobe™-1 has been stored for two years at room temperature in subdued light without detectable degradation as assessed by UV and HPLC analyses. Hypoxyprobe™-1 solutions in 0.9% saline have been stored at a concentration of 10 gms/liter (34 millimolar) at 4oC in subdued light for 1.5 years without detectable degradation (UV and HPLC analyses). When exposed to laboratory light, Hypoxyprobe™-1 slowly turns yellow.
Although doses of Hypoxyprobe™-1 up to 400 mg/kg have been used without measurable toxicity in mice (36), a dose of 60 mg/kg body weight is routinely used in studies of tissue hypoxia in rodents. The high water solubility of Hypoxyprobe™-1 permits small volume injections to be made which is convenient for studies with small animals. Intravenous injection or intraperitoneal injection can be used. The plasma half-life of Hypoxyprobe™-1 is 0.5 hours in C3H/He mice. Hypoxyprobe™-1 is distributed to all tissues in the body including brain but binds only to cells that have oxygen concentrations less than 14 micromolar which is equivalent to a pO2 of 10 mm Hg at 37oC. Tumors and normal liver, kidney and skin have cells at, or below, this pO2. For dogs, whole body doses of 0.28 gm/m2 are recommended (16). The plasma half-life for Hypoxyprobe™-1 in dogs is 1.5 ± 1.0 hours.
In addition to animal studies, Hypoxyprobe™ kits can be used for cells in tissue culture(6, 44). Typically, cell suspensions are incubated under hypoxia for 1 to 2 hours in the presence of 100 to 200 micromolar Hypoxyprobe™-1. The cells are harvested by cytospin, fixed and immunostained with an antibody for pimonidazole adducts. Sufficient concentrations of pimonidazole adducts are formed on the surface of cells to elicit a response to complement or activated cytotoxic lymphocytes (44).
The second part of the Hypoxyprobe™ Kits contains a primary antibody reagent that recognizes Hypoxyprobe adducts in hypoxic cells. NPI, Inc. offers three different primary reagents: a) a mouse monoclonal antibody (Hypoxyprobe™-1 Kit); b) a FITC conjugated mouse monoclonal antibody (Hypoxyprobe™-1 Plus Kit); and c) rabbit antisera (Hypoxyprobe™-1 Omni Kit). A detailed protocol for each antibody reagent is supplied as a part of a Product Insert that accompanies each shipment. NPI, Inc does not supply secondary antibody reagents as part of the Hypoxyprobe™-1 Kits but leaves Hypoxyprobe users to purchase standard secondary reagents that best meets their needs from third party suppliers.

a) Hypoxyprobe™-1 Kit. Hypoxyprobe™-1MAb1 is a monoclonal antibody IgG1 (MAb) that detects protein adducts of Hypoxyprobe™-1 in hypoxic cells. Hypoxyprobe™-1MAb1 is provided as an untreated exhausted supernatant from the hybridoma clone 4.3.11.3. The concentration of anti-Hypoxyprobe IgG in the supernatant is 70 micrograms/mL. Fifteen to ninety minutes after the injection of Hypoxyprobe™-1, tumor or normal tissue is excised or biopsied. (See FAQ for timing of tissue harvesting). The tissue of interest is then studied as frozen sections; formalin-fixed paraffin-embedded sections; or, as disaggregated tissues in flow cytometry assays. In the case of formalin-fixed, paraffin-embedded tissue sections, 150 uL of a 1:10 to 1:50 dilution of Hypoxyprobe™-1MAb1 is added to each tissue section. Chromogenic or fluorescent secondary antibody reagents are then used to reveal Hypoxyprobe™-1 adducts in the hypoxic tissue. For immunoperoxidase studies, we strongly recommend the use of the F(ab')2 secondary protocol (see below) because it can be used for a variety of animal species and tissues; it minimizes non-specific immunostaining; and, it is rapid. Hypoxyprobe™ kit HP-100 has enough hypoxia marker and antibody to analyze 5 sections from 35 mice. Hypoxyprobe™ kit HP-200 has enough hypoxia marker and monoclonal antibody to analyze 5 tissue sections from each of 133 mice (25 gram mice). Hypoxyprobe™ kit HP-1000 is designed for rat studies and has enough hypoxia marker and monoclonal antibody to analyze 10 tissue sections from each of 66 rats (250 gram rats).

b) Hypoxyprobe™-1 Plus kit. The Plus kit contains the Hypoxyprobe™-1MAb1 mouse monoclonal antibody IgG1 conjugated to FITC (four FITC residues per IgG1 molecule) at a concentration of 3 mg/ml. The antibody solution contains 0.15% sodium azide and is stored at 2-8oC. The Hypoxyprobe™-1 Plus Kit was designed for use on mouse tissues whereby the use of a secondary anti-FITC reagent obviates background problems often associated with the use of mouse monoclonal antibodies on mouse tissues. The FITC-conjugated mouse monoclonal is, therefore, an alternative to the use of the biotin conjugated F(ab)2 strategy with Hypoxyprobe™-1MAb1 described in the preceding paragraph.

c) Hypoxyprobe™-1 Omni Kit. The Omni kit contains PAb2627 rabbit antisera that has the advantage of minimizing background staining on tissues sections from all species except rabbit. It has been used successfully on human, dog, rat and mouse tissues. PAb2627 contains 0.15% sodium azide and is stored at 2-8o C.
 
Storage. Exhausted hybridoma supernatant containing Hypoxyprobe™-1MAb1 has been stored at = -20oC for 6 years without detectable loss of activity. For short term storage, undiluted Hypoxyprobe-1MAb1 supernatant containing 1 drop/mL of protein block (DAKO) or 10 mg/mL of bovine serum albumin and 10 millimolar sodium azide can be stored at 4oC for up to 4 months without detectable loss of activity.