TECHNICAL NOTE |
Correspondence to: Y. Otsuki, Dept. of Anatomy and Biology, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan.
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Summary |
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We describe an immunohistochemical method that allows the detection of apoptotic cells in human epidermis by use of confocal laser reflectance and antibodyimmunogoldsilver complexes. For this purpose, the site of free 3'-OH DNA ends was detected by the reflectance from heavy metal products (anti-digoxigenin antibodyimmunogoldsilver complexes) instead of 3, 3'-diaminobenzidine (DAB) reaction products in the conventional in situ nick end-labeling of DNA strand breaks (ISEL) technique. Localization of double-stranded DNA was demonstrated by the autofluorescence of methyl green. The ISEL technique using confocal reflectant laser microscopy (CRLM) clearly showed the most intense reflectance in the nuclei of granular cells, in contrast to only a weaker reflectance in those of basal cells. On the other hand, the extent of autofluorescence of methyl green was significantly more intense in the nuclei of basal cells and showed a reciprocal relation to that of the reflectance. Therefore, granular cells were most prone to apoptosis and did not contain double-stranded DNA, as indicated by the lack of stainability with methyl green. In addition, this method demonstrating the simultaneous localization of both free 3'-OH DNA ends and double-stranded DNA proved to have a wide range of applications, including the study of other DNA autolytic processes. (J Histochem Cytochem 46:783786, 1998)
Key Words: apoptosis, in situ DNA nick end-labeling technique, confocal reflectant laser microscopy, immunogoldsilver staining method, methyl green, autofluorescence
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Introduction |
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The ISEL TECHNIQUE is widely used and has the great advantage of allowing retrospective studies for the detection of apoptotic cells in archival material embedded in paraffin to be carried out (
In this study we modified the conventional ISEL technique to detect apoptotic cells in the human epidermis by using confocal reflectant laser microscopy (CRLM). Free 3'-OH DNA ends detected by the reflectance from the antibodyimmunogoldsilver complexes were present only in the nuclei of both the spinous and granulosa cells and not in the cytoplasm of the basal cells.
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Materials and Methods |
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Human scalp skin samples were embedded in Tissue Mount (Ciba Medical; Saitama, Japan) and rapidly frozen in acetone cooled on dry ice to prepare 67-µm-thick cryosections. Glass slides coated with aminopropyltriethoxysilane were used to eliminate background staining on slides. The sections were fixed in freshly prepared 10% (w/v) paraformaldehyde in 0.01 M PBS for 10 min and postfixed in a 2:1 mixture of ethanol and acetic acid for 5 min at -20C. After washing with PBS three times for 15 min, the sections were incubated with PBS containing 0.5% H2O2 for 20 min at room temperature (RT) to inhibit endogenous peroxidase activity. A commercially available kit (the ApopTag peroxidase kit ; Oncor, Gaithersburg, MD) was used for the detection of 3'-OH DNA ends in the sections. After washing with PBS for 15 min, the sections were soaked in the equilibration buffer of the kit for 1015 sec at RT and then incubated at 37C for 60 min in a moist chamber with 54 µl of the working buffer containing terminal deoxynucleotidyl transferase (TdT), digoxigenin-11-dUTP, and dATP. The reaction was stopped by incubating the sections in a blocking buffer containing Na-citrate and NaCl at 37C for 30 min. After rinsing with PBS three times for 15 min, the sections were used for the conventional ISEL or ISEL using CRLM study.
For the conventional ISEL study, some sections were incubated with an anti-digoxigenin antibody conjugated to horseradish peroxidase at RT for 30 min. After incubation with the antibody, the peroxidase activity was examined by exposing the sections to a solution containing 0.05% DAB and 0.01% H2O2 in Tris buffer, pH 7.6, for 36 min at RT. The sections were counterstained with 1% methyl green.
For the ISEL using CRLM study, the remaining sections were incubated with sheep anti-digoxigenin antibody conjugated to 10-nm colloidal gold (British BioCell International; Golden Gate, UK) at RT for 1 hr. After washing in distilled water, the sections were immersed in a physical developer containing silver lactate (Zymed; San Francisco, CA) at RT for 5 min. The reaction of the silver enhancement to the antibody labeled with immunogold particles was stopped by washing in distilled water. The sections were counterstained with 1% methyl green (pH 4.0) for 1 hr at RT. After dehydration with butanol, they were mounted in Entellan Neu (Merck; Darmstadt, Germany). The confocal laser microscope used in this study was an LSM-10 type (Carl Zeiss; Jena, Germany) equipped with two modes for fluorescence and reflectance. Both an argon laser excitation filter at 488 nm and an emission filter were used for detection of the autofluorescence of methyl green, which stains double-stranded DNA. The reflectance from the antibodyimmunogoldsilver complexes was detected using an argon laser excitation filter at 514 nm.
Negative controls were obtained by omitting either the antibody conjugates or the mixture of nucleotides and TdT.
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Results |
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This conventional ISEL study on the human epidermis demonstrated that DAB reaction was observed in both the nucleus and cytoplasm of keratinocytes. The nuclei of most granular cells exhibited intense DAB reaction, although those of spinous cells lacked the reaction. An intense DAB reaction in the cytoplasm was observed in the basal cells and, therefore, often made it difficult to differentiate between the reaction in the nuclei and that in the cytoplasm. Only a few spinous and granular cells exhibited a weak DAB reaction in the cytoplasm (Figure 1).
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The ISEL using CRLM study clearly showed that free 3'-OH DNA ends detected via the reflectance from the antibodyimmunogoldsilver complexes were present only in the nuclei of keratinocytes (Figure 2A). The intensity of the reflectance was different among all epidermal cell layers. The most intense reflectance was observed in the nuclei of granular cells. Moderately intense reflectance was detected in the nuclei of the spinous cells exhibiting a clustered staining pattern. The nuclei of most basal cells demonstrated only weak reflectance at the nuclear periphery. In contrast to the reflectance from antibodyimmunogoldsilver complexes, autofluorescence of methyl green, which stains double-stranded DNA, was detected in the nuceli of basal cells, but the granular cells lacked the autofluorescence or, if present, contained only weak autofluorescence (Figure 2B). The superimposed image of the reflectance and autofluorescence in the nuclei of spinous cells clearly demonstrated the different localization of the reflectance and autofluorescence. The reflectance was present at the nuclear periphery but the autofluorescence was demonstrated in the center of the nuclei (Figure 2C). The horny cells demonstrated neither reflectance nor autofluorescence (Figure 2AC).
No reflectance was observed in the controls.
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Discussion |
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Several studies have reported the localization of DNA fragmentation in normal human epidermis via the conventional ISEL technique (
Therefore, we applied the ISEL technique using antibodyimmunogoldsilver complexes instead of an antibodyimmunoperoxidase complex for detection of apoptotic cells containing free 3'-OH DNA ends in the human epidermis. The immunogoldsilver staining (IGSS) method is extensively employed for light, scanning electron, and transmission electron microscopy (
Methyl green has been known to stain double-stranded DNA (
Based on the above-mentioned findings, the ISEL using CRLM combined with methyl green staining has a wide range of applications, including the study of other DNA autolytic processes.
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Literature Cited |
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