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Immunogold electron microscopy using skin in michel’s medium intended for immunofluorescence analysis
Immunogold Electron Microscopy Using Skin in Michel’s Medium Intended for Immunofluorescence Analysis PAOLO SORELLI MATTHEW J. GRATIAN BALBIR S. BHOGAL JOHN A. MCGRATH
I
n the investigation of patients with autoimmune blistering skin diseases, immunogold electron microscopy has proved to be a useful approach in identifying target antigens. Such data are often important in establishing accurate subtyping of an individual’s disease and may be relevant to optimizing treatment. Several immunoelectron microscopy techniques have been developed, although many are time-consuming and require sophisticated equipment and trained personnel. In addition, most skin biopsies taken from patients with suspected autoimmune blistering skin diseases received by diagnostic laboratories arrive in transport medium intended for light microscopic immunofluorescence analysis. We have therefore developed a new, quick and reliable direct preembedding immunogold electron microscopy technique that uses, as its substrate, skin sent to the laboratory in Michel’s medium. The approach is illustrated in skin biopsies from patients with bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita. Identification of target antigens in autoimmune blistering skin diseases is helpful in establishing a precise diagnosis. Immunofluorescence analysis, particularly using salt-split skin techniques or dual-labeling confocal microscopy, may provide useful information in helping to classify an individual’s disease, but immunoelectron microscopy (IEM) remains the “gold standard” investigation.1,2 In recent years, colloidal gold has superseded peroxidase as the preferred marker for IEM because of its granular nature and high electron density. It has been From the Departments of Immunofluorescence and Cell and Molecular Pathology, St. John’s Institute of Dermatology, The Guy’s King’s College and St. Thomas’ Hospitals’ Medical School, St. Thomas’ Hospital, London, England, United Kingdom. Address correspondence to Dr. John McGrath, Department of Cell and Molecular Pathology, St. John’s Institute of Dermatology, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, England, UK.
© 2001 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
used successfully to label extracellular and intracellular antigens in both pre- and postembedding techniques.3,4 Existing methods, however, are time-consuming and require sophisticated equipment and a high level of technical expertise. Thus, IEM is not always a practical option for most diagnostic immunofluorescence laboratories. Indeed, most skin specimens sent to a diagnostic laboratory will arrive in a transport medium, such as Michel’s medium, which has previously been shown to be ideal for direct immunofluorescence studies.5,6 The aim of our study was to see if we could also use such skin samples as a possible substrate for IEM. In this report we describe a novel preembedding immunoelectron microscopy method that is performed on cryoprotected frozen sections and uses 1 nm colloidal gold in conjunction with silver staining enhancement. To illustrate the technique, we conducted a study to localize the site of antigen-antibody deposition in bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita.
Materials and Methods Skin Biopsies Biopsies of perilesional skin from patients with autoimmune subepidermal blistering skin diseases were received in Michel’s medium. Samples had been in the medium at room temperature for 3 to 5 days. Based on clinical diagnosis, the biopsies were from patients with bullous pemphigoid (n ⫽ 5), mucous membrane pemphigoid (n ⫽ 3), or epidermolysis bullosa acquisita (n ⫽ 5). The skin samples were removed from the Michel’s medium and a 2 mm3 piece was taken from each case and washed in phosphate-buffered saline for IEM processing. The rest of the sample was processed for routine direct immunofluorescence analysis including 1 M sodium chloride splitting of part of the skin sample, as described elsewhere.7 0738-081X/01/$–see front matter PII S0738-081X(00)00177-2
Clinics in Dermatology
Table 1. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Y
2001;19:638 – 641
IMMUNOGOLD ELECTRON MICROSCOPY
639
Step-by-Step Approach Used in This IEM Method
Cryoprotect 2 mm3 pieces of skin in 20% glycerol or dextran for 2 hours at 4°C. Embed in OCT and snap-freeze in liquid nitrogen. Cut 10 m thick sections onto coverslips, air dry, wash in phosphate buffered saline for 15 minutes at room temperature. Incubate sections in 1 nm gold conjugated anti-human IgG diluted 1:20 in phosphate buffered saline for 1 hour at 37°C. Wash sections for 30 minutes in phosphate-buffered saline and then fix in 10% glutaraldehyde for 10 minutes at room temperature. Wash in water for 5 minutes and silver enhance while observing under light microscope for approximately 10–12 minutes. Stop silver enhancement by immersion in distilled water once faint silver-black linear labeling of dermal-epidermal junction becomes apparent. Wash sections for 30 minutes in phosphate-buffered saline and then dehydrate through a graded series of alcohols: 10%, 30%, 50%, 75%, 90%, 100% (5 minutes in each). Embed sections using inverted resin-filled capsules (Fig 1A). Resin should be freshly prepared TAAB 812 premix resin (medium hardener) and specimens are polymerized for 16 hours at 60°C. Remove capsules from the coverslips by immersion in liquid nitrogen (Fig 1B). Examine embedded sections by light microscopy (while still in capsule) and select a suitable area for ultrathin sectioning. Cut ultrathin sections (⬃70 nm) on an ultramicrotome (Ultracut E) and collect on nickel/palladium grids. Counterstain with 2% uranyl acetate in 50% ethanol for 20 minutes followed by Reynolds lead citrate for 1 minute. Examine and photograph under a transmission electron microscope (JEOL 100CX).
IEM Method Full details of the preembedding IEM method developed and used in this study are shown in Table 1 and Fig 1.
Results Immunofluorescence In salt-split patients’ skin, direct immunofluorescence showed immunoglobulin deposition at the dermal-epidermal junction in all 13 cases studied. In the five cases of bullous pemphigoid, immunostaining was seen on the roof of the split skin (Fig 2A). In the three cases of mucous membrane pemphigoid, fluorescence labeling was detected on the dermal side of the split in two cases (Fig 2B) and to the roof and base of the split in the other
case. In all five cases of epidermolysis bullosa acquisita the fluorescent staining was present on the dermal side of the split skin (Fig 2C). In all samples the ultrastructural appearances of hemidesmosomes, lamina densa, and anchoring fibrils were well preserved; however, there was focal disruption of the plasma membrane. All cases of bullous pemphigoid showed gold particle labeling on hemidesmosomes (Fig 2D). In two cases the gold particles were also seen immediately below the hemidesmosomes within the upper lamina lucida. In mucous membrane pemphigoid, the gold particles were seen in the lamina lucida, immediately above the lamina densa region of the basement membrane zone (Fig 2E). In epidermolysis bullosa acquisita the gold particles labeled the anchoring fibrils beneath the lamina densa (Fig 2F).
Figure 1. Diagrammatic illustration of the inverted capsule technique used to transfer the cryostat skin section for electron microscopy processing. (A) A BEAM capsule (Agar Scientific Ltd, Essex, UK) is approximately 75% filled with medium hardness TAAB 812 premix resin (TAAB Laboratories Ltd, UK). The capsule is then inverted on top of the skin section on the coverslip and the resin is allowed to polymerize. (B) The coverslip and the capsule are then submerged in liquid nitrogen to detach the coverslip. The skin section separates within the resin and can be sectioned on an ultramicrotome.
Clinics in Dermatology
640 SORELLI ET AL
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2001;19:638 – 641
Figure 2. Direct split-skin immunofluorescence (A–C) and IEM (D–F) findings in the different immunobullous diseases studied. (A) In bullous pemphigoid, labeling is present at the roof of split skin. (B) In this case of mucous membrane pemphigoid, immunostaining is localized to the base of split skin. (C) In epidermolysis bullosa acquisita, immunoglobulin deposition is also present at the base of split skin. (D) In bullous pemphigoid, the immunogold labeling is noted overlying hemidesmosomes within the basal keratinocyte. (E) In mucous membrane pemphigoid, there is immunolabeling within the lower lamina lucida/lamina densa interface. (F) In epidermolysis bullosa acquisita, the gold particles are seen beneath the lamina densa in association with anchoring fibrils (magnifications, A–C ⫻25; D–F ⫻26,000).
Discussion The ideal IEM technique results in sensitive, uniform, and specific labeling with good preservation of tissue ultrastructure. Using the 1 nm immunogold silver staining method on frozen sections we have achieved these objectives. We have shown that the immunoreactants in bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita are localized to different structures in the basement membrane zone, thus enabling differentiation between these diseases. This method is able to distinguish between cases of mucous membrane pemphigoid and epidermolysis bullosa acquisita in which immunofluorescence analysis shows similar dermal labeling on split patients’ skin. (Compare Figs 2B and 2C with 2E and 2F). The main advantage of this method is that it utilizes skin sent to the laboratory in transport medium (ie, without the need for preplanning for IEM). In addition, the use of light microscopic silver enhancement means that only specimens with positive immunolabeling at this preliminary stage need be further processed for immunoultrastructural analysis. Also,
these findings help determine the optimal site for ultrathin sectioning. We believe that this modified preembedding IEM approach represents a useful additional practical option in selecting a technique to immunolocalize target antigens in autoimmune subepidermal blistering skin diseases.
References 1. Kazama T, Yamamoto Y, Hashimoto T, et al. Application of confocal microscopy to differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita. Br J Dermatol 1998;138:593– 601. 2. Woodley D, Saunders D, Talley M, et al. Localization of basement membrane components after dermal-epidermal junction separation. J Invest Dermatol 1983;81:149 –53. 3. Ishida-Yamamoto A, Eady RA, Watt FM, et al. Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis. J Histochem Cytochem 1996;44:167–75. 4. McGrath JA, Ishida-Yamamoto A, Shimizu H, et al. Immunoelectron microscopy of skin basement membrane zone antigens: A pre-embedding method using 1-nm immuno-
Clinics in Dermatology
Y
2001;19:638 – 641
gold with silver enhancement. J Acta Derm Venereol 1994; 74:197–200. 5. Vaughan Jones SA, Salas J, McGrath JA, et al. A retrospective analysis of tissue-fixed immunoreactants from skin biopsies maintained in Michel’s medium. Br J Dermatol 1994;189(Suppl 1):131–2. 6. Vaughan Jones SA, Palmer I, Bhogal BS, et al. The use of
IMMUNOGOLD ELECTRON MICROSCOPY
641
Michel’s transport media for immunofluorescence and immunoelectron microscopy in autoimmune bullous diseases. J Cutan Pathol 1995;22:365–70. 7. Bhogal B, Black MM. Diagnosis, diagnostic and research techniques. In: Wojnaroska F, Briggaman R, editors. Management of blistering diseases. London: Chapman and Hall Medical, 1990:15–34.
T
he Buffalo nickel, minted between 1913–1938, had two very American images on the obverse and reverse of the coin. The reverse has the familiar buffalo (American Bison) which was modeled after Black Diamond, a bison that actually lived at the New York Zoological gardens. The obverse is a composite of three American Indian Chiefs. During the depression, homeless men would carve into the nickel changing the image of the bison into an elephant or a donkey, then trading the created art piece for a meal or other necessity. These altered coins were known as “hobo nickels.”
REFERENCE: 1. Yeoman RS. A guide book of United States coins. 53rd ed. New York, NY: Whitman, 2000:109.
I
n the investigation of patients with autoimmune blistering skin diseases, immunogold electron microscopy has proved to be a useful approach in identifying target antigens. Such data are often important in establishing accurate subtyping of an individual’s disease and may be relevant to optimizing treatment. Several immunoelectron microscopy techniques have been developed, although many are time-consuming and require sophisticated equipment and trained personnel. In addition, most skin biopsies taken from patients with suspected autoimmune blistering skin diseases received by diagnostic laboratories arrive in transport medium intended for light microscopic immunofluorescence analysis. We have therefore developed a new, quick and reliable direct preembedding immunogold electron microscopy technique that uses, as its substrate, skin sent to the laboratory in Michel’s medium. The approach is illustrated in skin biopsies from patients with bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita. Identification of target antigens in autoimmune blistering skin diseases is helpful in establishing a precise diagnosis. Immunofluorescence analysis, particularly using salt-split skin techniques or dual-labeling confocal microscopy, may provide useful information in helping to classify an individual’s disease, but immunoelectron microscopy (IEM) remains the “gold standard” investigation.1,2 In recent years, colloidal gold has superseded peroxidase as the preferred marker for IEM because of its granular nature and high electron density. It has been From the Departments of Immunofluorescence and Cell and Molecular Pathology, St. John’s Institute of Dermatology, The Guy’s King’s College and St. Thomas’ Hospitals’ Medical School, St. Thomas’ Hospital, London, England, United Kingdom. Address correspondence to Dr. John McGrath, Department of Cell and Molecular Pathology, St. John’s Institute of Dermatology, St. Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, England, UK.
© 2001 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
used successfully to label extracellular and intracellular antigens in both pre- and postembedding techniques.3,4 Existing methods, however, are time-consuming and require sophisticated equipment and a high level of technical expertise. Thus, IEM is not always a practical option for most diagnostic immunofluorescence laboratories. Indeed, most skin specimens sent to a diagnostic laboratory will arrive in a transport medium, such as Michel’s medium, which has previously been shown to be ideal for direct immunofluorescence studies.5,6 The aim of our study was to see if we could also use such skin samples as a possible substrate for IEM. In this report we describe a novel preembedding immunoelectron microscopy method that is performed on cryoprotected frozen sections and uses 1 nm colloidal gold in conjunction with silver staining enhancement. To illustrate the technique, we conducted a study to localize the site of antigen-antibody deposition in bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita.
Materials and Methods Skin Biopsies Biopsies of perilesional skin from patients with autoimmune subepidermal blistering skin diseases were received in Michel’s medium. Samples had been in the medium at room temperature for 3 to 5 days. Based on clinical diagnosis, the biopsies were from patients with bullous pemphigoid (n ⫽ 5), mucous membrane pemphigoid (n ⫽ 3), or epidermolysis bullosa acquisita (n ⫽ 5). The skin samples were removed from the Michel’s medium and a 2 mm3 piece was taken from each case and washed in phosphate-buffered saline for IEM processing. The rest of the sample was processed for routine direct immunofluorescence analysis including 1 M sodium chloride splitting of part of the skin sample, as described elsewhere.7 0738-081X/01/$–see front matter PII S0738-081X(00)00177-2
Clinics in Dermatology
Table 1. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Y
2001;19:638 – 641
IMMUNOGOLD ELECTRON MICROSCOPY
639
Step-by-Step Approach Used in This IEM Method
Cryoprotect 2 mm3 pieces of skin in 20% glycerol or dextran for 2 hours at 4°C. Embed in OCT and snap-freeze in liquid nitrogen. Cut 10 m thick sections onto coverslips, air dry, wash in phosphate buffered saline for 15 minutes at room temperature. Incubate sections in 1 nm gold conjugated anti-human IgG diluted 1:20 in phosphate buffered saline for 1 hour at 37°C. Wash sections for 30 minutes in phosphate-buffered saline and then fix in 10% glutaraldehyde for 10 minutes at room temperature. Wash in water for 5 minutes and silver enhance while observing under light microscope for approximately 10–12 minutes. Stop silver enhancement by immersion in distilled water once faint silver-black linear labeling of dermal-epidermal junction becomes apparent. Wash sections for 30 minutes in phosphate-buffered saline and then dehydrate through a graded series of alcohols: 10%, 30%, 50%, 75%, 90%, 100% (5 minutes in each). Embed sections using inverted resin-filled capsules (Fig 1A). Resin should be freshly prepared TAAB 812 premix resin (medium hardener) and specimens are polymerized for 16 hours at 60°C. Remove capsules from the coverslips by immersion in liquid nitrogen (Fig 1B). Examine embedded sections by light microscopy (while still in capsule) and select a suitable area for ultrathin sectioning. Cut ultrathin sections (⬃70 nm) on an ultramicrotome (Ultracut E) and collect on nickel/palladium grids. Counterstain with 2% uranyl acetate in 50% ethanol for 20 minutes followed by Reynolds lead citrate for 1 minute. Examine and photograph under a transmission electron microscope (JEOL 100CX).
IEM Method Full details of the preembedding IEM method developed and used in this study are shown in Table 1 and Fig 1.
Results Immunofluorescence In salt-split patients’ skin, direct immunofluorescence showed immunoglobulin deposition at the dermal-epidermal junction in all 13 cases studied. In the five cases of bullous pemphigoid, immunostaining was seen on the roof of the split skin (Fig 2A). In the three cases of mucous membrane pemphigoid, fluorescence labeling was detected on the dermal side of the split in two cases (Fig 2B) and to the roof and base of the split in the other
case. In all five cases of epidermolysis bullosa acquisita the fluorescent staining was present on the dermal side of the split skin (Fig 2C). In all samples the ultrastructural appearances of hemidesmosomes, lamina densa, and anchoring fibrils were well preserved; however, there was focal disruption of the plasma membrane. All cases of bullous pemphigoid showed gold particle labeling on hemidesmosomes (Fig 2D). In two cases the gold particles were also seen immediately below the hemidesmosomes within the upper lamina lucida. In mucous membrane pemphigoid, the gold particles were seen in the lamina lucida, immediately above the lamina densa region of the basement membrane zone (Fig 2E). In epidermolysis bullosa acquisita the gold particles labeled the anchoring fibrils beneath the lamina densa (Fig 2F).
Figure 1. Diagrammatic illustration of the inverted capsule technique used to transfer the cryostat skin section for electron microscopy processing. (A) A BEAM capsule (Agar Scientific Ltd, Essex, UK) is approximately 75% filled with medium hardness TAAB 812 premix resin (TAAB Laboratories Ltd, UK). The capsule is then inverted on top of the skin section on the coverslip and the resin is allowed to polymerize. (B) The coverslip and the capsule are then submerged in liquid nitrogen to detach the coverslip. The skin section separates within the resin and can be sectioned on an ultramicrotome.
Clinics in Dermatology
640 SORELLI ET AL
Y
2001;19:638 – 641
Figure 2. Direct split-skin immunofluorescence (A–C) and IEM (D–F) findings in the different immunobullous diseases studied. (A) In bullous pemphigoid, labeling is present at the roof of split skin. (B) In this case of mucous membrane pemphigoid, immunostaining is localized to the base of split skin. (C) In epidermolysis bullosa acquisita, immunoglobulin deposition is also present at the base of split skin. (D) In bullous pemphigoid, the immunogold labeling is noted overlying hemidesmosomes within the basal keratinocyte. (E) In mucous membrane pemphigoid, there is immunolabeling within the lower lamina lucida/lamina densa interface. (F) In epidermolysis bullosa acquisita, the gold particles are seen beneath the lamina densa in association with anchoring fibrils (magnifications, A–C ⫻25; D–F ⫻26,000).
Discussion The ideal IEM technique results in sensitive, uniform, and specific labeling with good preservation of tissue ultrastructure. Using the 1 nm immunogold silver staining method on frozen sections we have achieved these objectives. We have shown that the immunoreactants in bullous pemphigoid, mucous membrane pemphigoid, and epidermolysis bullosa acquisita are localized to different structures in the basement membrane zone, thus enabling differentiation between these diseases. This method is able to distinguish between cases of mucous membrane pemphigoid and epidermolysis bullosa acquisita in which immunofluorescence analysis shows similar dermal labeling on split patients’ skin. (Compare Figs 2B and 2C with 2E and 2F). The main advantage of this method is that it utilizes skin sent to the laboratory in transport medium (ie, without the need for preplanning for IEM). In addition, the use of light microscopic silver enhancement means that only specimens with positive immunolabeling at this preliminary stage need be further processed for immunoultrastructural analysis. Also,
these findings help determine the optimal site for ultrathin sectioning. We believe that this modified preembedding IEM approach represents a useful additional practical option in selecting a technique to immunolocalize target antigens in autoimmune subepidermal blistering skin diseases.
References 1. Kazama T, Yamamoto Y, Hashimoto T, et al. Application of confocal microscopy to differential diagnosis of bullous pemphigoid and epidermolysis bullosa acquisita. Br J Dermatol 1998;138:593– 601. 2. Woodley D, Saunders D, Talley M, et al. Localization of basement membrane components after dermal-epidermal junction separation. J Invest Dermatol 1983;81:149 –53. 3. Ishida-Yamamoto A, Eady RA, Watt FM, et al. Immunoelectron microscopic analysis of cornified cell envelope formation in normal and psoriatic epidermis. J Histochem Cytochem 1996;44:167–75. 4. McGrath JA, Ishida-Yamamoto A, Shimizu H, et al. Immunoelectron microscopy of skin basement membrane zone antigens: A pre-embedding method using 1-nm immuno-
Clinics in Dermatology
Y
2001;19:638 – 641
gold with silver enhancement. J Acta Derm Venereol 1994; 74:197–200. 5. Vaughan Jones SA, Salas J, McGrath JA, et al. A retrospective analysis of tissue-fixed immunoreactants from skin biopsies maintained in Michel’s medium. Br J Dermatol 1994;189(Suppl 1):131–2. 6. Vaughan Jones SA, Palmer I, Bhogal BS, et al. The use of
IMMUNOGOLD ELECTRON MICROSCOPY
641
Michel’s transport media for immunofluorescence and immunoelectron microscopy in autoimmune bullous diseases. J Cutan Pathol 1995;22:365–70. 7. Bhogal B, Black MM. Diagnosis, diagnostic and research techniques. In: Wojnaroska F, Briggaman R, editors. Management of blistering diseases. London: Chapman and Hall Medical, 1990:15–34.
T
he Buffalo nickel, minted between 1913–1938, had two very American images on the obverse and reverse of the coin. The reverse has the familiar buffalo (American Bison) which was modeled after Black Diamond, a bison that actually lived at the New York Zoological gardens. The obverse is a composite of three American Indian Chiefs. During the depression, homeless men would carve into the nickel changing the image of the bison into an elephant or a donkey, then trading the created art piece for a meal or other necessity. These altered coins were known as “hobo nickels.”
REFERENCE: 1. Yeoman RS. A guide book of United States coins. 53rd ed. New York, NY: Whitman, 2000:109.