TECHNICAL NOTE |
Correspondence to: Jimmy Van heusden, Janssen Research Foundation, Dept of Oncology, Turnhoutseweg 30, B-2340 Beerse, Belgium.
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Summary |
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Immunocytochemical detection of bromodeoxyuridine (BrdU) labeling can be hampered by low BrdU incorporation levels. We describe here an amplification method for weak BrdU immunosignals. The tyramide signal amplification method based on catalyzed reporter deposition (CARD) uses fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The enzyme catalyzes the formation of highly reactive tyramide radicals with a very short half-life, resulting in the binding of fluorescein-conjugated tyramide only at the site of the enzymatic reaction. MCF-7 cells were grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented by growth factors. Under these culture conditions, the BrdU immunosignal was hard to detect but could be enhanced specifically by the tyramide signal amplification system, resulting in clear-cut differences between BrdU-negative and BrdU-positive cells. This enabled rapid and objective quantification of the BrdU labeling index without the risk of underestimating the number of cells in S-phase. Therefore, this amplification of BrdU immunosignals might also prove valuable for in vivo cancer prognosis, cell kinetics studies, and computer-assisted image analyses. (J Histochem Cytochem 45:315-319, 1997)
Key Words: BrdU, CARD, immunocytochemistry, MCF-7, signal amplification, tyramide
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Introduction |
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Assessment of cell proliferation is considered a basic parameter in cancer studies. A range of techniques have evolved to quantify this process (
However, the immunocytochemical detection of BrdU labeling can be hampered by low BrdU incorporation levels. Low incorporation levels of labeled nucleotides can occur as a consequence of altered cell growth rate (
Catalyzed reporter deposition (CARD) is a signal amplification method that has been described by
Although originally developed for ELISA, this technique has been adapted for immunohistochemistry on tissue sections (
In this study, MCF-7 human breast cancer cells were used, grown in vitro in medium containing charcoal-stripped fetal bovine serum supplemented with growth factors. Under these culture conditions, these cells are known to have an increased cell doubling time (
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Materials and Methods |
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Materials
Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/liter glucose, phenol red-free DMEM with 1 g/liter glucose and 1 mM sodium pyruvate, fetal bovine serum (FBS), L-glutamine, gentamicin, bovine insulin, basic fibroblast growth factor, sodium selenite, and transferrin were purchased from Life Technologies (Ghent, Belgium).
Dextran T500 was obtained from Pharmacia (Uppsala, Sweden) and activated charcoal from Sigma (St Louis, MO). Charcoal-stripped FBS was prepared by treating FBS with dextran-coated charcoal (DCC-FBS), according to the protocol of
Monoclonal antibody to BrdU (clone BU-1) containing nuclease activity was purchased from Amersham (Poole, UK). Secondary biotinylated goat anti-mouse IgG was obtained from DAKO A/S (Prosan; Ghent, Belgium) and secondary fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG from Southern Biotechnology Associates (Birmingham, AL). The Tyramide Signal Amplification (TSA)-Direct kit (Green) was obtained from DuPont NEN Products (Brussels, Belgium) and contained blocking reagent, horseradish peroxidase-conjugated streptavidin, 2 x concentrated amplification diluent, and fluorescein-labeled tyramide.
Cell Culture
Routinely, MCF-7 human mammary carcinoma cells, purchased from the American Type Culture Collection (Rockville, MD), were cultured in DMEM with 4.5 g/liter glucose supplemented by 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µg/ml gentamicin. The MCF-7 subclone used in this study has been described previously (
For the growth experiments, cells were cultured for 6 days in phenol red-free DMEM containing 5% DCC-FBS, 4.5 g/liter glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µg/ml gentamicin, 30 nM sodium selenite, and 10 µg/ml transferrin. Then cells were seeded onto Chamber Slides (Nunc; Naperville, IL), coated with 50 µg/ml poly-L-lysine (PLL) 1 day before use, at a concentration of 15,000 cells/chamber. Cells were allowed to attach for 24 hr and thereafter growth factors were added (10 µg/ml final concentration insulin and 5 ng/ml final concentration basic fibroblast growth factor). Cells were grown under these conditions for 7 days, with medium changes 3 and 6 days after seeding.
Control cells, growing in medium containing 10% untreated FBS, were seeded from stock cultures onto PLL-coated Chamber Slides at a concentration of 2500 cells/chamber. Cells were grown under these conditions for 7 days, with medium changes 3 and 6 days after seeding.
BrdU Labeling and Immunodetection
Cells grown for 7 days under the different culture conditions described above were incubated for 2.5 hr with an excess (100 µM) of BrdU at 37C in an incubator. Thereafter, cells were washed twice with serum-free culture medium and fixed in methanol (5 min, -2C) and acetone (10 sec, -2C), and air-dried. Cells were then ready to use for BrdU detection according to the following Protocols A or B.
Protocol A: BrdU Detection by Conventional Immunofluorescence Staining
Protocol B: BrdU Detection by Tyramide Signal Amplification
The BrdU labeling index, i.e. the percentage of cells in S-phase of the cell cycle, was determined by counting cells under a fluorescence microscope (Axiophot; Zeiss, Ober-kocher, Germany) with a dual filter set for simultaneous visualization of both fluorescein and PI. About 800 cells were counted twice for each test condition per experiment. Results are presented as mean ± SEM (n = 3).
Statistical Analysis
Data were analyzed with the two-tailed Mann-Whitney U-test using Stat View II software (Abacus Concepts; Berkeley, CA). Significance was defined at the level of p<0.05.
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Results |
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Control MCF-7 cells, grown in medium containing untreated FBS, showed a BrdU labeling index (Table 1) of 36.8 ± 1.3%, as described previously (
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In contrast, cells grown in medium containing charcoal-stripped FBS supplemented with growth factors showed low BrdU incorporation levels. This was reflected by weak BrdU immunosignals, which were hard to detect using the conventional immunofluorescence staining procedure (Figure 1a). BrdU-positive nuclei were stained faintly and showed a heterogeneous punctate staining pattern (Figure 1a, inset). The BrdU labeling index (Table 1) was 27.9 ± 0.6%, which was statistically different (p<0.05) from that of cells grown in medium containing untreated serum.
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The tyramide signal amplification method strongly enhanced the BrdU immunosignal (Figure 1b) of cells grown in medium containing charcoal-stripped serum. The nuclei were now stained intensely and the majority of the cells were homogeneously BrdU-positive (Figure 1b, inset), whereas some cells partially retained their original punctate staining (Figure 1b, inset). On immunofluorescence microscopy, no significant fading of the fluorescein signal was observed, in contrast to the conventional immunofluorescence staining preparations, most probably owing to the intense amplification of the immunosignal.
After visualization with the tyramide signal amplification method, a BrdU labeling index (Table 1) of 26.9 ± 0.9% was obtained which was not statistically different (p = 0.14) from the labeling index (Table 1) of 27.9 ± 0.6% that was obtained with normal immunofluorescence staining. This means that no additional cells became labeled after the tyramide signal amplification.
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Discussion |
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MCF-7 cells, grown in medium containing charcoal-stripped FBS supplemented by growth factors, showed a decreased BrdU labeling index compared to that of cells grown in medium containing untreated fetal bovine serum. This was not surprising because it is known that MCF-7 cells are estrogen-dependent (
As a consequence of these culture conditions low BrdU incorporation levels were observed, most probably due to an increased cell doubling time (
Therefore, an immunofluorescence amplification procedure using fluorescein-labeled tyramide, based on the principle of CARD, was developed for cell cultures to enhance specifically the BrdU immunosignals. In contrast to the weak, heterogeneous BrdU staining using the conventional immunofluorescence protocol, the tyramide signal amplification method resulted in a much clearer and homogeneous BrdU immunosignal in the majority of the cells. The difference between BrdU-positive and BrdU-negative cells became clear-cut, which made quantification rapid, easy, and objective without the risk of underestimating the number of cells in S-phase. Some cells stained intensely but partially retained their punctate staining pattern, which is indicative of the specificity of the tyramide signal amplification in that it is restricted to the immediate surroundings of the HRP deposits. The BrdU labeling index determined after tyramide signal amplification was the same as that determined after conventional immunofluorescence staining. This suggests that the tyramide signal amplification method is specific and that no additional cells became positive.
The tyramide signal amplification for low BrdU levels might also lend itself to providing more accurate results for prognosis after in vivo BrdU administration. Another application of the tyramide signal amplification might be in cell kinetics studies. For that purpose, it would be necessary to adapt the current fluorescent amplification procedure so that cells could be stained in suspension for flow cytometric analysis. Furthermore, the tyramide signal amplification method might help in the development of computer-assisted image analysis. For this application, it is necessary to obtain consistently clear and stable immunostaining, resulting in high-contrast images that facilitate the identification of labeled and unlabeled nuclei.
In conclusion, we have described an amplification method using CARD to amplify specifically weak BrdU immunosignals due to low BrdU incorporation levels. Amplification was achieved by the use of fluorescein-labeled tyramide as a substrate for horseradish peroxidase. The tyramide signal amplification method holds the promise to be of great value for in vivo cancer prognosis, cell kinetics studies, flow cytometry, and computer-assisted image analysis.
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Acknowledgments |
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We thank Lambert Leijssen and Hans Henderickx for the photographic layout.
Received for publication June 6, 1996; accepted October 14, 1996.
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