Hypoxyprobeâ„¢-1 Plus Kit for the Detection of Tissue ... - Millipore

Hypoxyprobe-1 Plus Kit
for the Detection of Tissue Hypoxia
100 mg Pimonidazole
500 uL Hypoxyprobe-1 Mab1 FITC-Labeled
500 uL Secondary anti-FITC Mab Labeled with HRP
Cat. No. HP2-100
FOR RESEARCH USE ONLY
Not for use in diagnostic procedures
USA & Canada
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1

Introduction
Oxygen gradients exist in normal and tumor tissue. These gradients affect gene
expression and are important in normal and pathological conditions. Until
recently it was difficult to measure these gradients at the cellular level; however,
with the development of 2-nitroimidazole hypoxia markers, it is now possible to
accurately measure oxygen gradient at the cellular level. There are a number of
ways that hypoxia markers can be detected. The immunohistochemical
technique is particularly attractive because oxygen gradients can be visualized
and compared with underlying hierarchical structures in tissues and with gene
expression on a cell-by-cell basis.
This insert describes the Hypoxyprobe system and its application to studies of
tissue hypoxia under both normal and pathological conditions. The hypoxia
marker that has received most attention in the Hypoxyprobe system of
markers is Hypoxyprobe-1, which is also known as pimonidazole
hydrochloride.
Hypoxyprobe-1 (pimonidazole hydrochloride)
The kit contains reagents for an improved two-step immunohistochemical
procedure for detecting hypoxia in rodent tissues using a primary fluorescein
(FITC)-conjugated mouse monoclonal antibody (Mab) directed against
pimonidazole protein adducts and a secondary mouse anti-FITC Mab conjugated
to horseradish peroxidase (HRP). These reagents produce slides that are
virtually free of the non-specific background staining that is frequently observed
when rodent antibodies are utilized to detect antigens in rodent tissues (see Ref
50: J. Histochem Cytochem 52:837-839, 2004)
1

Background
In 1976 , Varghese et al. reported that 14 C-labelled misonidazole formed adducts
in hypoxic cells in vitro and in vivo (1) . It was subsequently found that adducts
form with thiol groups in proteins, peptides and amino acids in a way that all
atoms of the ring and side-chain of the 2-nitroimidazole are retained (2-5) .
Hypoxia (pO2 < 10 mmHg) is required for binding but binding is not dependent
on the presence of specialized redox enzymes such as P450 nitroreductases.
Furthermore, wide variations in NADH and NADPH levels do not change the
oxygen dependence of binding (6, 7) .
Chapman et al. showed that the oxygen dependence of binding was fortuitously
close to that for radiation resistance and suggested that misonidazole binding
might be used as a hypoxia marker in solid tumors (8) . The clinical feasibility of
the hypoxia marker idea was demonstrated by means of autoradiographic
analyses of 3 H-misonidazole binding in a variety of human tumors (9) . While the
3
H-misonidazole approach had limited clinical utility, it spurred the
development of a variety of non-invasive assays for tissue hypoxia based on 2nitroimidazoles.
These included single photon electron capture tomography,
positron emission tomography, nuclear medicine analysis and magnetic
resonance spectroscopy of suitably labeled 2-nitroimidazole analogues (for
review see ref. (10) ).
During 19 F MRS investigations of tumor hypoxia with the hexafluorinated 2nitroimidazole,
CCI-103F, it became clear that an histological assessment of
hypoxia would be a useful complement to non-invasive assays (11-13) . This led to
the invention of the immunochemical hypoxia marker technique based on
monoclonal and polyclonal antibodies raised against protein adducts of
reductively activated 2-nitroimidazoles (14, 15) . Preclinical testing of immunochemical
reagents in spontaneous canine tumors showed that immunochemical
hypoxia markers would be useful in their own right (16-21) . In addition to
providing a quantitative measure of hypoxia, immunohistochemical markers
provide insights into microregional relationships between hypoxia and factors
such as necrosis, proliferation, differentiation, apoptosis, and oxygen regulated
protein expression. A variety of immunochemical hypoxia markers have now
been used in clinical (22-27) and preclinical studies (16, 28-33) of such relationships.
An example of the unique value of the immunohistochemical marker approach
is the observation that neither metallothionein nor vascular endothelial growth
factor are expressed in the majority of hypoxic cells in human squamous cell
carcinomas (24, 25) even though in vitro studies would have predicted otherwise
(34, 35)
.
With respect to quantifying hypoxia by the immunochemical technique, image
analysis (24-27) or flow cytometric analysis (36, 37) appear most promising.
Preclinical studies of sampling error showed that stratification of patients is
2

feasible with the immunohistochemical approach if 3-4 biopsies are obtained
from geographically separate regions of each tumor. Precision can be increased
by increasing the number of sections analyzed per biopsy from 1 to 3 but
analysis of multiple biopsies is the most important factor. Interestingly, the
accuracy of the immunochemical analysis increases as the amount of hypoxia
decreases (19,21) .
Protein adducts of reductively-activated pimonidazole are effective immunogens
for the production of both polyclonal and monoclonal antibodies. The antibodies
have been used for immunoperoxidase analysis of formalin-fixed, paraffinembedded
sections (6, 16, 23-26, 32, 40) ; for immunofluorescence analysis of frozen
fixed sections (27, 41-43) ; and, for flow cytometry with directly labeled or
secondary fluorescent antibodies (36) .The antibodies have also been used in
enzyme linked immunosorbent assays (6, 16, 40) . One final attractive feature of
pimonidazole is the fact that pimonidazole adducts in vivo are long-lived (16) .
This provides flexibility in the timing of biopsy taking which is an advantage in
a clinical setting. In summary Hypoxyprobe-1 and associated antibodies form
a very attractive basis on which to develop a low tech, low cost kit for
measuring normal and tumor tissue hypoxia.
Application
The rationale for developing pimonidazole hydrochloride (Hypoxyprobe-1) as
a hypoxia marker for experimental and clinical use was based on its chemical
stability, water solubility, wide tissue distribution and, in the case of clinical
studies, the fact that human toxicity data were available from earlier
radiosensitizer trials. This facilitated early clinical application and
Hypoxyprobe kits have now been used in many experimental studies and
clinical trials worldwide. Solid Hypoxyprobe-1 is very stable being
unchanged after storage for 2 years at room temperature in subdued light. Saline
solutions of Hypoxyprobe-1 used for clinical studies (34 millimolar in 0.9%
saline, pH 3.9 ± 0.1) are extremely stable being unchanged after 1.5 years at 4 o C
in subdued light as determined by high performance liquid chromatography and
ultraviolet spectroscopy. In addition to chemical stability, Hypoxyprobe-1 has
high water solubility (400 millimolar; 116 grams per 1000 mL) that facilitates
intravenous marker infusion and produces a short plasma half-life of 5.1 ± 0.8
hours.
In spite of the water solubility of its hydrochloride salt (pKa 8.7), pimonidazole
itself has an octanol-water partition coefficient of 8.5 (38) and diffuses readily
into tumors and normal tissues including brain (39) . Consistent with a large, 155
liter volume of distribution, pimonidazole concentrates approximately 3 fold
above plasma levels in tumors and normal tissues (39) thereby increasing the
sensitivity of hypoxia marking. At the Hypoxyprobe-1 dose of 0.5 g/m 2 used
3

in hypoxia marking, pimonidazole causes neither central nervous system toxicity
nor sensation (e.g., flushing) (39). .Central nervous system toxicity was of
particular interest because this was the dose limiting toxicity for
Hypoxyprobe-1 at the higher, multiple doses used in radiosensitizer trials. In
addition to the absence of central nervous system effects, the overall procedure
from Hypoxyprobe-1 infusion to tumor biopsy is well-tolerated in both
inpatient and outpatient settings.
Currently, rodent (mouse and rat) animal models are frequently used to study
hypoxia in normal and tumor tissues, including human-to-rodent
xenotransplantation experiments. Because the Hypoxyprobe-1 Mab1, which
recognizes pimonidazole adducts in hypoxic tissue proteins, is of mouse origin,
the presence of normal mouse or highly homologous rat immunoglobulins
makes it difficult for the secondary antibody reagent to selectively bind to
Mab1. Therefore, even when special blocking procedures are employed, the
immunostaining of rodent tissues with Mab1 may result in a strong non-specific
background staining that can obscure the specific binding pattern of this primary
antibody. The Hypoxyprobe-1 Plus Kit for the Detection of Tissue Hypoxia
was developed to address this obstacle and ensure fast (two-step) and accurate
detection of hypoxia gradients in mouse and rat animal models. In this detection
system, the primary monoclonal antibody Mab1 is directly labeled with FITC,
while the secondary Mab (directed against FITC) is labeled with HRP. Since the
secondary Mab does not cross-react with antigen epitopes present in rodent
tissues, no non-specific background staining is generated using this set of
reagents.
For Research Use Only; Not for use in diagnostic procedures
4

Kit Components
1. Hypoxyprobe-1: (Part No.: 90203) One vial containing 100 mg of
Pimonidazole Hydrochloride
2. Hypoxyprobe-1 Mab1: (Part No.: 90531) One vial containing 500 µL of
purified Mab1 (clone 4.3.11.3) conjugated with FITC.
3. Anti-FITC secondary Mab: (Part No.: 90532) One vial containing 500 µL
of purified Mab against FITC (clone 5D6.2) conjugated with HRP.
Storage
The FITC-labeled Hypoxyprobe 1-Mab1 and the HRP-labeled Secondary Anti-
FITC monoclonal antibody can be stored at 2-8°C until the expiration date
indicated on the label. Keep dark.
Hypoxyprobe-1 (Pimonidazole Hydrochloride) has great chemical stability in
solid form and in aqueous solutions and requires no stabilizer. 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 g/liter (34 millimolar) at 4°C in subdued light for 1.5 years without
detectable degradation (UV and HPLC analyses). When exposed to laboratory
light, Hypoxyprobe-1 slowly turns yellow.
Assay Instructions
1. 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 37 o C. Tumors and normal liver, kidney
and skin have cells at, or below, this pO2. For dogs, whole body doses of
0.28 gm/m 2 are recommended (16) . The plasma half-life for Hypoxyprobe-
1 in dogs is 1.5 ± 1.0 hours.
5

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) . Hypoxyprobe-1
Plus Kit has enough hypoxia marker to administer to 67 (25 gram) mice or
6-7 (250 gram) rats.
2. The primary antibody reagent is a conjugate consisting of Hypoxyprobe-
1 MAb1 (mouse IgG1) covalently bound to FITC and is intended to detect
protein adducts of Hypoxyprobe-1 in hypoxic cells. The conjugate is
provided as a purified preparation at a concentration of 1 mg/mL. Fifteen to
ninety minutes after the injection of Hypoxyprobe-1, tumor or normal
tissue is excised or biopsied. 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, paraffinembedded
tissue sections, 100 µL of a 1:50 dilution of the conjugate is
added to each tissue section. Hypoxyprobe-1 Plus Kit has enough
primary conjugate to analyze 250 tissue sections at a 1:50 dilution. Optimal
working dilutions must be determined by the end user.
3. The secondary antibody reagent is a conjugate consisting of a mouse anti-
FITC covalently bound to HRP and is intended to detect Mab1-FITC
stained structures in target slides. The conjugate is provided as a purified
preparation at a concentration of 1 mg/mL. In the case of formalin-fixed,
paraffin-embedded tissue sections, 100 µL of a 1:50 dilution of the
conjugate is added to each tissue section. We recommend utilizing standard
colorimetric systems used in visualizing HRP-stained tissue sections in
routine IHC protocols. Hypoxyprobe-1 Plus Kit has enough secondary
conjugate to analyze 250 tissue sections at a 1:50 dilution. Optimal
working dilutions must be determined by the end user.
6

Peroxidase Immunohistochemistry Technique
Recommended procedure for immunostaining Hypoxyprobe-1 adducts in
formalin-fixed, paraffin-embedded tissues from a variety of animal species
including mice and rats. Other tissue fixation methods will require
optimization of the suggested protocol.
Improved immunohistochemical method for detecting hypoxia gradients in
mouse tissues and tumors. Samoszuk MK, Walter J, Mechetner E. J. Histochem.
Cytochem. 52(6):837-839, 2004.
Step
1
2
3
4
5
6
7
8
Procedure
Heat Fix tissue
slides
Deparaffinize
tissue
Deparaffinize
tissue
Deparaffinize
tissue
Clear slides of
Xylene
Clear slides of
Xylene
Begin hydrating
tissue
Continue to
hydrate
Time,
min.
Temp.
7
Reagents
60 60 o C None 1
3 RT Xylene 2
3 RT Xylene
3 RT Xylene
1 RT 100% Ethanol
1 RT 100% Ethanol
1 RT 70% Aqueous ethanol
1 RT 30% Aqueous ethanol
9 Hydrate tissue 1 RT Deionized water
10 Hydrate tissue 1 RT Deionized water
11
Transfer slides to
rinse buffer
2 RT 1x TBS + Tween 20 3
Notes

Step
12
Procedure
Quench tissue
peroxidase
Time,
min.
5 RT
Temp.
13 Antigen retrieval 20 Steamer
14
15
16
Transfer slides to
rinse buffer
Block non-specific
binding
Incubate in Primary
conjugate
8
Reagents
3% H2O2 in distilled
water
10mM Citrate, pH
6.0
2 RT 1x TBS + Tween 20 3
5 RT Blocking reagent 7
30 RT
Hypoxyprobe-1
MAb1 conjugated
with FITC (1:50)
17 Wash in rinse buffer 2 RT 1x TBS + Tween 20 3
18
Incubate in
Secondary conjugate
30 RT
Anti-FITC Mab
conjugated with
HRP (1:50)
19 Wash in rinse buffer 2 RT 1x TBS + Tween 20 3
20
Peroxidase
chromogen
10 RT DAB 10
21 Stop DAB reaction 1 RT Distilled water
22 Wash in rinse buffer 1 RT 1x TBS + Tween 20 3
23 Counterstaining 1 RT Hematoxylin 11
24 Wash in rinse buffer 1 RT 1x TBS + Tween 20 3
25
Dehydration &
Clearing
See
notes RT
100% alcohol and
Xylene
26 Coverslip N/a RT Permount 13
Notes
4
8
9
12

Technical Notes
1. This step may be performed in microwave oven on high for 2 minutes. If
the sections are still wet, the water will turn to steam and separate the tissue
from slides. Caution, glass can get very hot.
2. Per xylene incubation use clean xylene. Clear-Rite 3 is a non-toxic
alternative to xylene and is available from Richard-Allan Scientific,
Kalamazoo, MI (Cat. No. 6901). If using this alternative, increase the
incubation periods accordingly.
3. 20x TBS is Tris buffered saline, 10 mM (Chemicon Cat. No. 20845). All
rinses were done with 1x TBS working solution.
4. Make sure to rinse off the 3% H2O2 since it can interfere with results. Store
brand hydrogen peroxide may be utilized.
5. Antigen retrieval solution is 10x Citrate Buffer (Chemicon Cat. No. 21545)
used at a 1x working solution.
6. HIER method is at the end users discretion.
7. Blocking reagent (Chemicon Cat. No. 20773). 1% BSA or 1% normal
mouse serum in TBS may also be used.
8. The primary antibody reagent is a conjugate between Hypoxyprobe-1
MAb1 (mouse IgG1) and FITC. The conjugate is provided as a purified
preparation at a concentration of 1 mg/ml. Dilute the primary conjugate
solution 1:50 in Antibody Diluent (Chemicon Cat. No. 21544). Typically,
100 µL of diluted primary conjugate solution is applied to each tissue
section.
9. The secondary antibody reagent is a conjugate between a monoclonal
antibody against FITC and HRP provided as a purified preparation at a
concentration of 1 mg/mL. Dilute the secondary antibody solution 1:50 in
Antibody Diluent (Chemicon Cat. No. 21544). Typically, 100 µL of
diluted secondary conjugate is added to each tissue section.
10. Liquid 3,3'-diaminobenzidine reagent (DAB). DAB Chromogen-A
(Chemicon Cat. No. 71895) and DAB Chromogen-B (Chemicon Cat. No.
71896) mix at 1:25 ratio respectively.
11. Hematoxylin counter staining reagent (Chemicon Cat. No. 20844).
12. Use clean and fresh solutions to incubate slides for 1 minute in 4 changes of
100% alcohol and then to three changes of clean xylene.
13. Permount solution is available from Fisher Scientific (Cat. No. SP15-500).
9

Frequently Asked Questions
Q: What is the best solvent for Hypoxyprobe-1?
A: Hypoxyprobe-1 is the hydrochloride salt of a weak base and, as such, is
very soluble in aqueous solutions including neutral buffered saline (116
mg/mL or 400 millimolar).
Q. What dose of Hypoxyprobe-1 should be used for hypoxia marking
experiments?
A: For small animals of uniform size such as laboratory rats and mice, a dose
of Hypoxprobe-1 of 60 mg/kg body weight is recommended. Doses
ranging from 30 mg/kg (47) to 400 mg/kg (36) have been used in mice and rats
without toxicity or altered oxygen levels due to blood flow effects.
Nevertheless, significant effects on blood flow at doses above 100 mg/kg of
Hypoxyprobe-1 have been observed in tumors implanted in the hind legs
of mice. Caution must be taken, therefore, when doses > 100 mg/kg are used.
For larger animals with non-uniform body size, the dose is calculated on the
basis of surface area. For humans, the recommended dose is 0.5 gm/m2 (23)
while for dogs a dose of 0.28 gm/m2 has been used (16) .
Q. Does Hypoxyprobe-1 penetrate hypoxic brain and brain tumor
tissue?
A: Although Hypoxyprobe-1 is water soluble, its corresponding free base
has an octanol water coefficient of 8.5 and, as a result, the marker freely
penetrates into both brain and brain tumor tissue (48, 49) .
10

Q. Is Hypoxyprobe-1 the best probe for detecting hypoxia in vivo?
A: Although other markers are available for detecting hypoxia in vivo,
Hypoxyprobe-1 has some real advantages. Foremost is its high solubility in
aqueous solution (400 millimolar; 116 mg/mL of saline), which allows the
marker to be administered to rodents as small volume (= 0.1 mL) injections of
saline either intraperitoneally or intravenously. Other markers such as the
hexafluorinated CCI-103F have aqueous solubilities of 10 millimolar or less.
Although Hypoxyprobe-1, the hydrochloride salt of pimonidazole, is very
water soluble, pimonidazole itself has an octanol-water partition coefficient of
8.5 and penetrates all tissues including brain.
Another advantage is that pimonidazole binding can be detected by
immunofluorescence in frozen fixed tissue sections; by immunoperoxidase in
formalin-fixed paraffin-embedded tissue sections; by ELISA or by flow
cytometry.
Q. Can the monoclonal antibody to Hypoxyprobe-1 adducts be used on
mouse or rat tissues?
A: Yes. The described procedure results in a clean IHC staining that is free of
non-specific background caused by mouse or rat immunoglobulins
impregnating the surrounding tissue (50) .
11

References
1. Varghese, A. J., Gulyas, S., and Mohindra, J. K. (1976). Hypoxia-dependent
reduction of 1-(2-nitro-1-imidazolyl)-3-methoxy-2- propanol by Chinese hamster
ovary cells and KHT tumor cells in vitro and in vivo, Cancer Res. 36:3761-3765.
2. Chacon, E., Morrow, C. J., Leon, A. A., Born, J. L., and Smith, B. R. (1988).
Regioselective formation of a misonidazole-glutathione conjugate as a function of
pH during chemical reduction., Biochem. Pharmacol. 37:361-363.
3. Raleigh, J. A., Franko, A. J., Koch, C. J., and Born, J. L. (1985). Binding of
misonidazole to hypoxic cells in monolayer and spheroid culture: evidence that a
side-chain label is bound as efficiently as a ring label. Br. J. Cancer. 51:229-235.
4. Raleigh, J. A. and Koch, C. J. (1990). Importance of thiols in the reductive
binding of 2-nitroimidazoles to macromolecules. Biochem Pharmacol. 40:2457-
2464.
5. Varghese, A. J. (1983). Glutathione conjugates of misonidazole. Biochem Biophys
Res Commun. 112:1013-1020.
6. Arteel, G. E., Thurman, R. G., Yates, J. M., and Raleigh, J. A. (1995). Evidence that
hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde
perfusion of rat liver. Br J Cancer. 72:889-895.
7. Arteel, G. E., Thurman, R. G., and Raleigh, J. A. (1988). Reductive metabolism of
the hypoxia marker pimonidazole is regulated by oxygen tension independent of the
pyridine nucleotide redox state. Eur. J. Biochem. 253:743-750.
8. Chapman, J. D., Franko, A. J., and Sharplin, J. A (1981). marker for hypoxic cells
in tumours with potential clinical applicability. Br. J. Cancer. 43:546-550.
9. Urtasun, R. C., Koch, C. J., Franko, A. J., Raleigh, J. A., and Chapman, J. D.
(1986). A novel technique for measuring human tissue pO2 at the cellular level. Br.
J. Cancer. 54:453-457.
10. Raleigh, J. A., Dewhirst, M. W., and Thrall, D. E. Measuring tumor hypoxia., Sem.
Radiat. Oncol. 6:37-45, 1996.
11. Jin, G. Y., Li, S. J., Moulder, J. E., and Raleigh, J. A. Dynamic measurements of
hexafluoromisonidazole (CCI-103F) retention in mouse tumours by 1H/19F
magnetic resonance spectroscopy, Int. J. Radiat. Biol. 58: 1025-1034, 1990.
12. Raleigh, J. A., Franko, A. J., Treiber, E. O., Lunt, J. A., and Allen, P. S. Covalent
binding of a fluorinated 2-nitroimidazole to EMT-6 tumors in BALB/C mice:
Detection by F-19 nuclear magnetic resonance at 2.35T, Int. J. Radiat. Oncol. Biol.
Phys. 12: 1243-1245, 1986.
12

13. Raleigh, J., Franko, A., Kelly, D., Trimble, L., and Allen, P. (1991). Development
of an in vivo 19F magnetic resonance method for measuring oxygen deficiency in
tumors. Magn. Res. Med. 22:451-466.
14. Raleigh, J. A., Miller, G. G., Franko, A. J., Koch, C. J., Fuciarelli, A. F., and
Kelly, D. A. (1987). Fluorescence immunohistochemical detection of hypoxic cells
in spheroids and tumours. Br J Cancer 56:395-400.
15. Raleigh, J. A., Miller, G. G., Franko, A. J., and Chapman, J. D. Immunochemical
detection of hypoxia in normal and tumor tissue. USA patent 5,086,068, February,
1992.
16. Azuma, C., Raleigh, J. A., and Thrall, D. E. (1997). Longevity of pimonidazole
adducts in spontaneous canine tumors as an estimate of hypoxic cell lifetime.
Radiat. Res. 148:35-42.
17. Cline, J. M., Thrall, D. E., Page, R. L., Franko, A. J., and Raleigh, J. A. (1990).
Immunohistochemical detection of a hypoxia marker in spontaneous canine
tumours. Br. J. Cancer 62:925-931.
18. Cline, J. M., Thrall, D. E., Rosner, G. L., and Raleigh, J. A. (1994). Distribution
of the hypoxia marker CCI-103F in canine tumors. Int J Radiat Oncol Biol Phys.
28:921-933.
19. Cline, J., Rosner, G., Raleigh, J., and Thrall, D. (1997). Quantification of CCI-
103F labeling heterogeneity in canine solid tumors. Int. J. Radiat. Oncol. Biol.
Phys. 37:655-662.
20. Thrall, D. E., McEntee, M. C., Cline, J. M., and Raleigh, J. A. (1994). ELISA
quantification of CCI-103F binding in canine tumors prior to and during
irradiation. Int. J. Radiat. Oncol. Biol. Phys. 28:649-659.
21. Thrall, D. E., Rosner, G. L., Azuma, C., McEntee, M. C., and Raleigh, J. A.
(1997). Hypoxia marker labeling in tumor biopsies: quantification of labeling
variation and criteria for biopsy sectioning. Radiother Oncol. 44:171-176.
22. Evans, S. M., Hahn, S., Pook, D. R., Jenkins, W. T., Chalian, A. A., Zhang, P.,
Stevens, C., Weber, R., Weinstein, G., Benjamin, I., Mirza, N., Morgan, M.,
Rubin, S., McKenna, W. G., Lord, E. M., and Koch, C. J. (2000). Detection of
hypoxia in human squamous cell carcinoma by EF5 binding. Cancer Res. 60:
2018-2024.
23. Kennedy, A. S., Raleigh, J. A., Perez, G. M., Calkins, D. P., Thrall, D. E.,
Novotny, D. B., and Varia, M. A. (1997). Proliferation and hypoxia in human
squamous cell carcinoma of the cervix: first report of combined
immunohistochemical assays. Int J Radiat Oncol Biol Phys. 37:897-905..
13

24. Raleigh, J. A., Calkins-Adams, D. P., Rinker, L. H., Ballenger, C. A., Weissler,
M. C., Fowler, W. C., Jr., Novotny, D. B., and Varia, M. A. (1998). Hypoxia and
vascular endothelial growth factor expression in human squamous cell carcinomas
using pimonidazole as a hypoxia marker. Cancer Res. 58:3765-3768.
25. Raleigh, J., Chou, S.-C., Calkins-Adams, D., Ballenger, C., Rinker, L., Novotny,
D., and Varia, M. (2000). A clinical study of hypoxia and metallothionein protein
expression in squamous cell carcinomas. Cl. Cancer Res. 6:855-862.
26. Varia, M. A., Calkins-Adams, D. P., Rinker, L. H., Kennedy, A. S., Novotny, D.
B., Fowler, W. C., Jr., and Raleigh, J. A. (1998). Pimonidazole : a novel hypoxia
marker for complementary study of tumor hypoxia and cell proliferation in
cervical carcinoma. Gynecol. Oncol. 71:270-277.
27. Wijffels, K., Kaanders, J., Rijken, P., Bussink, J., Van den Hoogen, F., Marres,
H., de Wilde, P., Raleigh, J., and Van der Kogel, A. (2000). Vascular architecture
and hypoxic profiles in human head and neck squamous cell carcinomas. Br. J.
Cancer. 83:674-683.
28. Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K.,
Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C.
J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., and Keshet, E. (1998). Role of
HIF-1alpha in hypoxia-mediated apoptosis, cell proliferation and tumour
angiogenesis. Nature 394:485-490.
29. Hodgkiss, R. J. (1998). Use of 2-nitroimidazoles as bioreductive markers for
tumour hypoxia. Anticancer Drug Des. 13:687-702.
30. Koch, C. J., Chasan, J. E., Jenkins, W. T., Chan, C. Y., Laughlin, K. M., and
Evans, S. M. (1998). Co-localization of hypoxia and apoptosis in irradiated and
untreated HCT116 human colon carcinoma xenografts. Adv Exp Med Biol. 454:
611-618..
31. Raleigh, J. A., Zeman, E. M., Calkins, D. P., McEntee, M. C., and Thrall, D. E.
(1995). Distribution of hypoxia and proliferation associated markers in spontaneous
canine tumors. Acta Oncol. 34:345-349.
32. Raleigh, J. A., Chou, S.-C., Tables, L., Suchindran, S., Varia, M. A., and Horsman,
M. R. (1998). Relationship of hypoxia to metallothionein expression in murine
tumors. Int. J. Radiat. Oncol. Biol. Phys. 42:727-730.
33. Waleh, N. S., Brody, M. D., Knapp, M. A., Mendonca, H. L., Lord, E. M., Koch, C.
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