TECHNICAL NOTE |
Correspondence to: Akira Ishiko, Dept. of Dermatology, Keio Univ. School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan..
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Summary |
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Cryoultramicrotomy was originally established to provide ultrathin cryosections as substrates for on-section immunolabeling in immunoelectron microscopy. Recently, we recognized that ultrathin cryosections of skin (0.2 µm thick) could serve as substrates for immunofluorescence (IF) with excellent resolution. To assess the advantages and the limitations of IF on ultrathin cryosections, we compared the labeling of IF on 0.2-µm ultrathin cryosections of skin with those of routine IF on 6-µm cryostat sections, confocal laser scanning microscopy (LSM), and immunogold electron microscopy using several markers of keratinocyte cell surface and basement membrane zone molecules. IF on ultrathin cryosections clearly demonstrated a lack of bullous pemphigoid antigens beneath the melanocytes, desmosomal antigens as discontinuous dot-like labeling, and nondesmosomal plasma membrane antigen as a ladder-like pattern. IF on ultrathin cryosections provided convincing images with higher resolution than confocal LSM, which corresponded well to those of immunogold electron microscopy. IF on ultrathin cryosections had superior resolution compared to routine IF or confocal LSM and should serve as a powerful tool in future studies for the analysis of skin antigens. (J Histochem Cytochem 46:14551460, 1998)
Key Words: immunohistochemistry, basement membrane zone, desmosome, hemidesmosome, confocal laser scanning microscopy, cryoultramicrotomy
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Introduction |
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Cryoultramicrotomy was first introduced for immunoelectron microscopy (immuno-EM) to enable ultrathin sectioning at very low temperatures without embedding in plastic resin (
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Materials and Methods |
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Antibodies
For the BMZ markers, the following antibodies were used: two bullous pemphigoid (BP) sera that bound only to the 230-kD BP antigen (BPAG1) or the 180-kD BP antigen (BPAG2) by immunoblot as described previously (6 integrin (
Ultrathin Cryosections (0.10.2 µm Thick)
Ultrathin cryosections of normal human skin were obtained basically according to a method described by Tokuyasu (
Routine Cryostat Sections (6 µm Thick)
Normal human skin was immersed in OCT compound and rapidly frozen by isopentane cooled with liquid nitrogen. Then 6-µm sections were cut using a routine cryostat microtome at -25C.
Immunofluorescence
Immunofluorescence was performed on both routine 6-µm cryostat sections and 0.2-µm ultrathin cryosections of normal human skin, as follows. The sections were incubated with primary antibodies at 37C for 1 hr, washed with PBS, and incubated with FITC-conjugated secondary antibodies (DAKOPATTS; Copenhagen, Denmark) at 37C for 1 hr. The immunolabeled sections were mounted with glycerol/PBS containing p-phenylenediamine as an antifading agent (
Confocal Laser Scanning Microscopy
Skin cryosections (6 µm thick) were attached to the glass slides. The specimens were incubated with primary antibodies, followed by FITC-conjugated secondary antibodies as described above. The sections were mounted with glycerol/PBS containing p-phenylenediamine and observed under confocal LSM (MRC-600; Nippon Bio-Rad Laboratory, Tokyo, Japan) as described (
Immunoelectron Microscopy
Immunogold-EM was performed using either the postembedding method (
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Results |
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IF on Ultrathin Cryosections Provided Superior Resolution of BMZ Antigens Compared to Confocal LSM and Routine IF on 6-µm Sections
All the BMZ antigens of BPAG1, BPAG2, 6 integrin, Type VII collagen, and Type IV collagen were observed linearly at the dermoepidermal junction with IF on 0.2-µm cryoultrathin sections (Figure 1a, Figure 1d, and Figure 1g), confocal LSM (Figure 1b, Figure 1e, and Figure 1h), and routine IF on 6-µm sections (Figure 1c, Figure 1f, and Figure 1i). However, IF on ultrathin cryosections provided superior resolution, i.e., the immunolabeling was observed as sharp, thin, linear signaling, and fine microvilli of basilar surface of basal keratinocyte were clearly demonstrated on the 0.2-µm ultrathin cryosections (Figure 1a, Figure 1d, and Figure 1g). Furthermore, the lack of the BPAG1 and the BPAG2 (Figure 1d) beneath the melanocytes was observed as occasional discontinuous labeling, while Type VII collagen and Type IV collagen showed a continuous pattern (data not shown). The labeling of
6 integrin, another marker for hemidesmosomes, was indistinguishable from that of BP antigen by IF on ultrathin cryosections (Figure 1g).
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Confocal LSM had better resolution compared to routine IF and revealed labeling patterns similar to those of cryoultrathin sections (Figure 1b, Figure 1e, and Figure 1h). However, fluorescence was occasionally detected as a blurred line at the dermoepidermal junction, and the results were less convincing than those of IF on ultrathin cryosections, which provided sharper and thinner signaling, thus enabling the tracing of fine microvilli.
Immunogold EM showed distinct distributions of BPAG1, BPAG2, and 6 integrin as described previously, i.e., BPAG1 was localized in the intracellular portion of hemidesmosomes, BPAG2 was localized along the plasma membrane of hemidesmosomes (
6 integrin was localized on the extracellular surface of hemidesmosomes (data not shown). These differences in ultrastructural localization of antigens could not be detected even by IF on ultrathin cryosections or confocal LSM, indicating the limitation of immunofluorescence.
IF on Ultrathin Cryosections Provided Superior Resolution of Keratinocyte Cell Surface Antigens and Could Distinguish Desmosomal Antigens from Nondesmosomal Antigens
By routine immunofluorescence, all the antibodies to keratinocyte cell surface antigens used in this study, including PF (against Dsg1) (Figure 2c) and PV (against Dsg3) (not shown) sera, anti-desmoplakin antibodies (Figure 2f), and anti-desmoyokin antibodies (Figure 2i), labeled continuously on the keratinocyte cell surface and no significant difference was seen among them. By confocal LSM, all immunolabeling, including desmoyokin, appeared as a discontinuous dot-like pattern with no significant difference among the antibodies (Figure 2b, Figure 2e, and Figure 2h). Using 0.2-µm ultrathin cryosections as substrate, however, the labeling was seen to follow two patterns. The first was a discontinuous dot-like pattern shown by PF serum (Figure 2a), PV serum (not shown), and anti-desmoplakin monoclonal antibody (Figure 2d). PF and PV sera could not be distinguished by this method, but desmoplakin was labeled as larger dots than PF or PV antigens. This dot-like pattern may represent the desmosomal localization of antigens, as demonstrated below by immuno-EM. The second was a ladder-like pattern in the desmoyokin labeling in which the signals were seen along the plasma membrane of keratinocytes (Figure 2g). This pattern was specific to desmoyokin, which localized at the nondesmosomal plasma membrane of keratinocyte as demonstrated by immuno-EM.
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In immuno-EM (data not shown), PF antigen and PV antigen were mainly localized at the extracellular portion of desmosome and were indistinguishable from each other, as described previously (
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Discussion |
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In this study, we demonstrated that IF on 0.2-µm ultrathin cryosections of skin can provide better resolution than confocal LSM or routine 6-µm cryostat sections. IF on routine sections has been widely used for antigen immunolocalization at the light microscopic level. Confocal LSM was recently introduced to improve the rejection of out-of-focus noise of IF and to greatly enhance images. On the other hand, improved techniques for immuno-EM have been established for investigating the ultrastructural localization of antigens. Typically, immunohistochemistry using light microscopy and immuno-EM are performed on different tissue preparations, and there may be a gap in the findings between them. Ultrathin skin cryosections from a single block can serve as substrate for both IF and immunogold EM simultaneously, and correlative microscopy by immunofluorescence and immunogold EM on the same block is feasible. For example, a lack of BP antigens beneath the melanocyte, which was visible by immunoperoxidase EM (
Confocal LSM is known to have superior resolution compared to conventional IF, and the improvements are such that it is possible to optically section thick specimens with little degradation in the image quality of interior sections. One can theoretically derive an expression for the thickness of the confocal sections for the limiting case of an infinitely small confocal aperture as follows (
(where = wavelength of light = 0.488 µm, n = index of refraction for the immersion medium = 1.5, and NA = numerical aperture = 1.3).
The theoretical axial resolution of confocal LSM is calculated as follows:
The axial resolution of confocal LSM is apparently thicker than 0.2 µm. Therefore, the rejection of out-of-focus noise is theoretically considered to be better in IF on ultrathin cryosections than in confocal LSM. Additional merits of the present method are that apparatus for confocal LSM is not necessary, and that, once cut, the ultrathin cryosections can be stored for at least 6 months at -30C until use. Moreover, on the surface immuno-EM can be conducted on the sections from the same block of tissue after the IF on ultrathin cryosections. The disadvantages of this method include the need for apparatus for cryoultramicrotomy and the fact that skillful sectioning is required.
The dot-like staining at the keratinocyte cell surface observed with PV antigen, PF antigen, and desmoplakin can be interpreted to represent desmosomal localization of these antigens in combination with immuno-EM. On the other hand, the ladder-like pattern seen with desmoyokin can be interpreted to represent the antigen localization of keratinocyte plasma membrane without desmosomal attachment plaques. The present method can therefore be used for differentiation of desmosomal antigen from nondesmosomal antigen without immuno-EM.
In conclusion, IF on ultrathin cryosections, with its resolution superior to that of confocal LSM, provides a better understanding of molecular distribution in combination with immuno-EM labeling, and therefore serves as a powerful new tool for investigating immunolocalization of skin antigens.
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Acknowledgments |
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Supported by Grants-in-Aid for Scientific Research (Nos. 05404036 and 07457191) from the Ministry of Education, Science, and Culture of Japan, and by Keio Gijuku Academic Development Funds.
We thank Ms Megumi Sato for excellent technical help. We are grateful to Dr D.R. Garrod (University of Manchester), Dr I. M. Leigh (London Hospital, UK), Dr T. Hashimoto (Kurume University, Japan), and Dr A. Sonnenberg (University of Amsterdam, The Netherlands) for providing antibodies.
Received for publication June 9, 1998; accepted August 25, 1998.
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