Journal of Histochemistry and Cytochemistry, Vol. 46, 783-786, June 1998, Copyright © 1998, The Histochemical Society, Inc.


TECHNICAL NOTE

Localization of Apoptotic Cells in the Human Epidermis by an In Situ DNA Nick End-labeling Method Using Confocal Reflectant Laser Microscopy

Y. Itoa and Y. Otsukia
a Department of Anatomy and Biology, Osaka Medical College, Osaka, Japan

Correspondence to: Y. Otsuki, Dept. of Anatomy and Biology, Osaka Medical College, 2-7, Daigaku-machi, Takatsuki, Osaka 569-8686, Japan.


  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 antibody–immunogold–silver complexes. For this purpose, the site of free 3'-OH DNA ends was detected by the reflectance from heavy metal products (anti-digoxigenin antibody–immunogold–silver 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 46:783–786, 1998)

Key Words: apoptosis, in situ DNA nick end-labeling technique, confocal reflectant laser microscopy, immunogold–silver staining method, methyl green, autofluorescence


  Introduction
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Introduction
Materials and Methods
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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 (Gavrieli et al. 1992 ; Ansari et al. 1993 ; Gold et al. 1994 ). In the conventional ISEL technique, the reaction products of DAB oxidized by horseradish peroxidase demonstrate the localization of 3'-OH DNA ends in apoptotic cells as a brown color (DAB reaction). In epidermis, melanosomes with peroxidase activity are present in the cytoplasm of basal cells. It is known empirically that the basal cells show a false-positive DAB reaction when they are incubated with a solution containing DAB. Therefore, the conventional ISEL technique involving immunoperoxidase method is unsuitable for study of the epidermis.

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 antibody–immunogold–silver complexes were present only in the nuclei of both the spinous and granulosa cells and not in the cytoplasm of the basal cells.


  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 6–7-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 10–15 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 3–6 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 antibody–immunogold–silver 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.


  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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Figure 1. Conventional ISEL technique demonstrates the intense DAB reaction in both the nuclei of the granular cells (arrows) and the cytoplasm of the basal cells (arrowheads). Bar = 27 µm.

Figure 2. Images of free 3'-OH DNA ends detected by confocal laser reflectance from antibody–immunogold–silver complexes and double-stranded DNA stained with methyl green. (A) Intense reflectance is detected in the nuclei of granular cells, in contrast to only weak reflectance in the basal cells. (B) The intense autofluorescence of methyl green was detected in the nuclei of basal cells, whereas the nuclei of granular cells lacked autofluorescence. (C) The superimposed images of A and B clearly demonstrate the different intensity of the reflectance (red) and autofluorescence (green) among the layers of human epidermis. B, basal cells; S, spinous cells; G, granular cells.

The ISEL using CRLM study clearly showed that free 3'-OH DNA ends detected via the reflectance from the antibody–immunogold–silver 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 antibody–immunogold–silver 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 2A–C).

No reflectance was observed in the controls.


  Discussion
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Materials and Methods
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Several studies have reported the localization of DNA fragmentation in normal human epidermis via the conventional ISEL technique (Gavrieli et al. 1992 ; Polakowska et al. 1994 ; Tamada et al. 1994 ), but there has been much discussion concerning which cell in the epidermis is most susceptible to treatment with the conventional ISEL technique, only granular cells (Gavrieli et al. 1992 ; Tamada et al. 1994 ) or both the granular and spinous cells (Polakowska et al. 1994 ). In the present study using the conventional ISEL technique, we demonstrated an intense DAB reaction in the nuclei of granular cells and in the cytoplasm of basal cells in the human epidermis. Our findings indicate that all keratinocytes possessing melanosomes with peroxidase activity in the cytoplasm react positively when the conventional ISEL technique with immunoperoxidase is used, regardless of the degree of DNA strand breaks.

Therefore, we applied the ISEL technique using antibody–immunogold–silver complexes instead of an antibody–immunoperoxidase complex for detection of apoptotic cells containing free 3'-OH DNA ends in the human epidermis. The immunogold–silver staining (IGSS) method is extensively employed for light, scanning electron, and transmission electron microscopy (Otsuki and Maxwell 1993 ) and exhibits a higher labeling sensitivity (more than fourfold) than does the indirect immunoperoxidase staining method (Scopsi et al. 1986 ; Hayat 1993 ). The use of CRLM combined with the IGSS method revealed weak reflectance in some nuclei of basal cells. This may be due to DNA strand breaks induced by DNA repair systems or to the site of active gene transcription located in the euchromatin (Thiry 1991 ; Migheli et al. 1995 ), rather than to oligonucleosomal degradation and background.

Methyl green has been known to stain double-stranded DNA (Franklin and Filion 1981 ; Melnick and Pickering 1988 ; Burres et al. 1993) and is weakly autofluorescent (Rothbarth et al. 1976 ). The ISEL using CRLM technique combined with methyl green staining enables the simultaneous localization of both 3'-OH DNA ends and double-stranded DNA in apoptotic cells and also the comparison of their concentrations. In this study, the superimposed image of both the reflectance from the antibody–immunogold–silver complexes and the autofluorescence of methyl green clearly demonstrated that apoptotic change involving the newly formed 3'-OH DNA ends began in the spinous cells and was typical in granular cells that lacked the double-stranded DNA. This topographical relationship between 3'-OH DNA ends and double-stranded DNA of keratinocytes is supported by the results of a biochemical study carried out by Suzuki et al. 1977 in which the distribution pattern of DNA in keratinocytes changed during keratinization, resulting in a lack of double-stranded DNA in all electron-opaque areas of the nuclei in the granular cells.

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.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Ansari B, Coates PJ, Greenstein BD, Hall PA (1993) In situ end-labeling detects DNA strand breaks in apoptosis and other physiological and pathological states. J Pathol 170:1-8[Medline]

Burres NS, Frigo A, Rasmussen RR, McAlpine JB (1992) A colorimetric microassay for the detection of agents that interact with DNA. J Nat Prod 55:1582-1587[Medline]

Franklin AL, Filion WG (1981) Acridine orange-methyl green fluorescent staining of nucleoli. Stain Technol 56:343-348[Medline]

Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragments. J Cell Biol 119:493-501[Abstract]

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Hayat MA (1993) Immunogold-silver staining overview. J Histotechnol 16:197-199

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Otsuki Y, Maxwell LE (1993) Immunogold-silver staining method of lymphocyte cell surface antigens with light, transmission electron, and scanning electron microscopy. J Histotechnol 16:217-221

Polakowska RR, Piacentini M, Bartlet R, Goldsmith LA, Haake AR (1994) Apoptosis in human skin development: morphogenesis, periderm and stem cells. Dev Dyn 199:176-188[Medline]

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Suzuki H, Fukuyama K, Epstein WL (1977) Changes in nuclear DNA and RNA during epidermal keratinization. Cell Tissue Res 184:155-167[Medline]

Tamada Y, Tamada H, Kitamura T, Yokochi K, Nitta Y, Ikeya T, Matsumoto Y (1994) Identification of programmed cell death in normal human skin tissues by using specific labeling of fragmented DNA. Br J Dermatol 131:521-524[Medline]

Thiry M (1991) In situ nick translation at the electron microscopic level: a tool for studying the location of DNase-sensitive regions within the cell. J Histochem Cytochem 39:871-874[Abstract]