TECHNICAL NOTE |
Correspondence to: Ralph J. Panos, Pulmonary Div., Northwestern U. Medical School, Passavant 777, 303 E. Superior St., Chicago, IL 60611.
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Summary |
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Incorporation of halogenated nucleotide analogues is often used to assess DNA synthesis and to quantitate cellular proliferation. Multiple antibodies have been developed to bromodeoxyuridine (BrdUrd) and it is the most frequently utilized substrate. Because the immunodetection of incorporated BrdUrd requires DNA denaturation or nuclease digestion, most of these antibodies are not reactive in tissues or cells fixed with crosslinking agents. Antigen retrieval techniques utilizing protease digestion restore BrdUrd antigenicity and permit the detection of BrdUrd in formalin-fixed tissue. However, during the development of a double label immunohistochemical protocol to quantitate proliferating alveolar Type II cells, we noted nucleus-specific staining in lung sections from animals that had not received BrdUrd. Therefore, we systematically analyzed the specificity of the immunohistochemical detection of incorporated BrdUrd in formalin-fixed tissue after protease digestion. Enzymatic antigen recovery diminished the specificity of the BrdUrd reaction product and caused false-positive staining with the BU-1, B44, and BR3 monoclonal antibodies. Staining was less prominent with Bu20a but was more specific. Protease antigen recovery may decrease the specificity of BrdUrd immunodetection. Appropriate controls are required when enzymatic digestion is used to detect incorporated BrdUrd in formalin-fixed tissue. The type and duration of fixation, antibody to BrdUrd, protease, and tissue may affect the specificity of the staining pattern. (J Histochem Cytochem 45:1165-1170, 1997)
Key Words: proliferation, bromodeoxyuridine, protease antigen recovery
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Introduction |
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Bromodeoxyuridine (BrdUrd) is a halogenated pyrimidine that may substitute for thymidine in newly synthesized DNA. Various monoclonal antibodies (MAbs) to BrdUrd have been developed and used in immunoassays to detect cell proliferation, cell cycle kinetics, sister chromatid exchange, and to isolate nascent DNA (
In the development of a double label immunohistochemical staining protocol to quantitate proliferating alveolar Type II cells, we found that antigen retrieval with several different proteases decreased the specificity of various MAbs for BrdUrd (
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Materials and Methods |
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Male Sprague-Dawley rats (225-250 g) were injected IP with 100 mg/kg BrdUrd dissolved in sterile, endotoxin-free PBS at a concentration of 10 mg/ml. Eighteen hr later the animals were sacrificed and the testes removed and fixed in 10% phosphate-buffered formalin. The testes were then embedded in paraffin and 4-µm sections were mounted on 3-aminopropyltriethoxysilane coated slides to enhance adherence. After air-drying, the slides were heated overnight at 50C. They were dewaxed in xylene and rehydrated through a graded series of ethanol solutions (100%, 100%, 70%, 50% v/v) and finally washed in PBS. Endogenous peroxidase activity was inhibited by treating the slides with 5% H2O2 in methanol twice for 5 min. The sections were then washed in PBS and permeabilized by incubating in 0.1% Triton X-100 dissolved in PBS for 30 min at room temperature (RT). After washing in PBS, the sections were incubated with the various proteases under these conditions: pepsin (Sigma; St Louis, MO), 0.1% solution (w/v) dissolved in 0.1 N HCl for 30 min at 37C; pronase E (Sigma), 100 µg/ml dissolved in 0.02 M Tris-HCl, pH 7.6, containing 20 mM CaCl2-2·H2O for 30 min at 37C; protease Type XXIV (Sigma), 0.1% solution (w/v) dissolved in 0.1 M phosphate buffer, pH 7.4, for 15 min at RT; trypsin Type III (Sigma), 0.1% solution (w/v) dissolved in 0.05 M Tris-HCl, pH 7.6, containing 0.1% CaCl2 2· H2O for 30 min at 37C. For the experiments determining the effect of the duration of fixation, the concentrations of the enzymes were increased to 0.2% (w/v) pepsin, 500 µg/ml pronase E, 0.2% (w/v) protease Type XXIV, and 0.2% (w/v) trypsin Type III. The slides were then washed in PBS and nuclear histones were extracted from DNA by incubating the sections in cold 0.1 N HCl for 10 min. Double-stranded DNA was denatured with 2 N HCl for 30 min at 37C. [This step was omitted for sections exposed to the BU-1 antibody (Amersham; Arlington Heights, IL), which was applied with a proprietary mixture of nucleases.] HCl was neutralized with 0.1 M borax, pH 8.5, for 10 min at RT. After washing in PBS, the sections were incubated with 3% horse serum in PBS for 30 min at RT. Excess blocking solution was blotted off and primary antibodies applied under the following conditions. BU-1 (Amersham) proprietary antibody/nuclease solution was applied undiluted for 1 hr at RT. B44 (Becton-Dickson; San Jose, CA) was applied at a 1:4 dilution (6.25 µg/ml) in 3% horse serum containing 0.1% Triton X-100 overnight at 4C. BR3 (Caltag Laboratories; So. San Francisco, CA) was applied at a 1:200 dilution (5.0 µg/ml) ) in 3% horse serum containing 0.1% Triton X-100 overnight at 4C. Bu20a (Dako; Carpinteria, CA) was applied at a 1:50 dilution (6.0 µg/ml) ) in 3% horse serum containing 0.1% Triton X-100 overnight at 4C. After incubation with the primary antibody, the sections were washed in PBS twice for 5 min each.
The sections were then incubated with biotinylated horse anti-mouse (heavy and light chain-specific) IgG (Vector Laboratories; Burlingame, CA) diluted 1:200 in 3% horse serum/0.1% Triton X-100 for 1 hr at RT and then washed in two changes of PBS for 5 min each. Biotin-streptavidin-horseradish peroxidase diluted 1:100 in PBS was added for 30 min at RT. After washing in two changes of 0.1 M phosphate buffer, pH 7.4, the horseradish peroxidase activity was detected using Ni/Co enhancement of the diaminobenzidine reaction product (
All sections were coded and independently graded by both investigators, who rated the slides on a scale of 0 to 5: 0, no staining; 1, sporadic nuclear staining in basal cells; 2, nuclear staining in all basal cells; 3, staining in basal cells throughout the section and extending to the intermediate cells intermittently; 4, staining in basal cells and extending to the intermediate cells throughout the section; 5, nuclear staining throughout the section. Data are expressed as the mean ± SEM of three separate experiments. Statistical analysis was performed using an unpaired t-test (StatView; Abacus Concepts) and p<0.05 was considered significant.
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Results |
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Antigen recovery with the different enzymes significantly affected the detection of BrdUrd (Figure 1). With both the B44 and BR-3 anti-BrdUrd MAbs, nuclear staining was present in unlabeled tissues that were not treated with proteases as well as in the enzymatically digested specimens. There were no significant differences in the labeling scores between specimens from BrdUrd-treated and control animals. No nuclear labeling was present in tissues from saline-instilled rats using the BU-1 MAb and either no antigen recovery or trypsin treatment. Pronase E, protease, and pepsin digestion produced equivalent labeling scores in BrdUrd-labeled and unlabeled specimens.
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Bu20a was the most specific MAb and accurately discriminated BrdUrd-labeled tissues from unlabeled sections after either no treatment, pronase E, or trypsin digestion. However, after protease or pepsin antigen recovery, there were no significant differences between BrdUrd-treated and control animals.
To determine the effect of fixation duration on Bu20a detection of incorporated BrdUrd, tissue was fixed for 31-510 hr in buffered formalin (Figure 2). After 510 hr of fixation, no staining was detected in untreated sections, whereas at earlier time points Bu20a accurately discriminated between labeled and unlabeled tissues. Nuclear staining was present in unlabeled tissues with all the enzymatic treatments except trypsin. The highest levels of nonspecific staining occurred with protease and pepsin antigen recovery (Figure 3).
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Discussion |
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Many MAbs to BrdUrd have been developed. A halogenated ribouracil conjugated to a carrier protein has been the immunogen used in the creation of many of these antibodies. They are generally of low affinity (Ka approximately 106 M-1 for BrdUrd in solution) and detect the halogenated nucleotide only in single-stranded but not in double-stranded DNA (
We utilized protease antigen recovery in the development of a double label immunohistochemical technique to quantitate proliferating alveolar Type II cells (
The cause of the false-positive staining is uncertain. Other investigators have suggested that enzymatic digestion may unmask or create antigenic epitopes common to unrelated tissue constituents (
In a study comparing BrdUrd with [3H]-thymidine labeling in spermatagonial stem cells and Leydig cells in the testes and small intestinal crypt cells, Thoolen (
Immunohistochemical detection of BrdUrd has been widely used to determine cell proliferation in cells and tissues. Enzymatic antigen recovery allows BrdUrd immunodetection in tissues preserved with aldehyde-containing fixatives. Careful and appropriate controls are required when protease digestion is used to determine a proliferative index based on BrdUrd immunodetection in formalin-fixed tissue specimens.
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Acknowledgments |
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This work was funded by the Department of Veterans Affairs.
Received for publication December 10, 1996; accepted December 30, 1996.
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