RAPID COMMUNICATION |
Correspondence to: Nagahito Saito, Dept. of Gastroenterology & Hematology, Hokkaido University Graduate School of Medicine, Kita-15 jo, Nishi-7 chome, Kita-ku, Sapporo, Japan. E-mail: nagahitosaito@k7.dion.ne.jp
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Summary |
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A novel antigen retrieval procedure was carried out in the post-embedding immunogold electron microscopy method to improve the stainability of the samples. This was done by weakly fixing cultured Helicobacter pylori (ATCC43504) and embedding in Lowicryl K4M. Before staining with the anti-H. pylori antibody, the ultrathin sections were mounted on a nickel grid and heated at 121C for 15 min, 99C for 40 min, and 65C for 24 hr in distilled water, 0.1 M phosphate buffer (pH 7.4), 0.01 M EDTA (pH 7.2), 0.05 M Tris buffer (pH 10.0), 0.8 M urea (pH 7.2), 0.01 M citric acid (pH 6.0), or a commercially available target unmasking fluid (S1699; pH 6.0). Antigen retrieval in the Tris buffer solution generally showed better stainability than the classical post-embedding method without any antigen retrieval. At 65C for 24 hr, better stainability of the ultrasections was observed for each of the solutions used except for the phosphate buffer compared to the control. We suggest that the antigen retrieval method should be applied for routine use even by in post-embedding immunogold electron microscopy. (J Histochem Cytochem 51:989994, 2003)
Key Words: antigen retrieval, immunogold staining, post-embedding method, electron microscopy, Tris buffer, EDTA, H. pylori, Lowicryl K4M
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Introduction |
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Examination of the ultrastructural localization of intracellular protein is important for investigating cell function. This is generally done by the immunogold staining method using pre- or post-embedding. From our experience, frozen sections produced by immunocryoultramicrotomy are the most suitable for immunolabeling by the pre-embedding method (
About a decade ago, an antigen retrieval (AR) technique, which made the various antigens that had been difficult to stain in formalinparaffin sections detectable, was introduced. It described the process of heating tissues using a microwave oven (
In this study we used apparatus that is different from a microwave oven to overcome the heating problems induced by a microwave oven and statistically compared the efficiency of the AR under several heating conditions. We used Helicobacter pylori as material for AR in our experiment (
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Materials and Methods |
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Bacterial Culture
The H. pylori strain (ATCC 43504), preserved at -80C, was inoculated and cultured in Brucella broth medium (DIFCO; Detroit, MI) with the addition of 10% heat-inactivated horse serum at 37C under microaerobic conditions (CO2 10%, O2 5%, N2 85%) with rolling for 24 hr.
Immunoelectron Microscopy
The bacteria were fixed in a mixture of 0.5% glutaraldehyde and 2% paraformaldehyde (PFA) in Hepes buffer (pH 6.8) for 15 min and then in 2% PFA in Hepes buffer (pH 6.8) for 14 days at 4C.
Because we attempted to elucidate the pathogenicity of H. pylori to human gastric cells, we tried to find the best fixative for both the bacteria and the adhered gastric cells. Therefore, a weaker fixative for preservation of the ultrastructure and antigenicity was needed. In a preliminary experiment, both the cultured H. pylori and the biopsied specimens from a human stomach were fixed with 2% PFA for 15 min, 4% PFA for 15 min followed by 2% PFA for 14 days, 0.2% glutaraldehyde (GA) plus 2% PFA for 15 min followed by 2% PFA for 14 days, 0.5% GA plus 2% PFA for 15 min followed by 2% PFA for 14 days, 0.5% GA plus 4% PFA for 30 min, and 1% GA plus 2% PFA for 30 min. These samples were then stained with some polyclonal antibodies without any AR. We concluded that fixation with 0.5% GA plus 2% PFA for 15 min followed by 2% PFA for 14 days was the most suitable for the post-embedding method. It preserved both the cell's antigenicity and ultrastructure in a biopsy specimen from human gut. This was demonstrated by an immunostaining experiment to examine the changes in the intracellular proteins and to observe the cell-to-cell contact area.
After dehydration with 80% ethanol, the specimens were infiltrated with increasing concentrations of Lowicryl K4M (Polysciences; Tokyo, Japan). Polymerization of Lowicryl K4M was performed by UV irradiation (wavelength peak at 360 nm) for 24 hr as previously reported (
Antigen Retrieval
After being sunk in 100% ethanol for 3 min, grids with ultrathin sections were transferred in Beem capsules and immersed in either (a) distilled water (DW), (b) 0.1 M phosphate buffer (PB, pH 7.4), (c) 0.01 M EDTA (pH 7.2), (d) 0.05 M Tris buffer (Tris, pH 10.0), (e) 0.8 M urea (urea, pH 7.2), (f) 0.01 M citric acid (CA, pH 6.0), or (g) a commercially available target unmasking fluid (pH 6.0, S1699; DAKO Japan, Tokyo, Japan) and then kept at either 121C for 15 min using an autoclave (BS-305; TOMY, Tokyo, Japan), at 99C for 40 min using a gene amplificator (PerkinElmer Gene Amp PCR system 2400; Norwalk, CT), or at 65C for 24 hr using a constant temperature box (SAKURA WAX-MELT; SAKURA Seiki, Tokyo, Japan).
Immunostaining was performed as follows. The grids were immersed in a mixture of 0.5% normal goat serum and 0.05% bovine serum albumin in 0.02 M phosphate buffer (pH 7.2) for 3 min and incubated in rabbit IgG fraction specific for H. pylori (DAKO Japan; diluted 1:200) for 4 hr, followed by reacting with 10-nm gold particle-labeled goat antiserum specific for rabbit IgG (GAR G10; Amersham, Arlington Heights, IL, diluted 1:10) for 1 hr. In the most effective solution, the AR effect was also compared at high, neutral, and low pH. As controls, the sections were stained without any heating soon after being sunk in 100% ethanol for 3 min (
To determine the efficiency of each staining after the various conditions of AR, the gold particles per 100 granules were counted. Because the degree of antigenicity of the flagella differed among the bacteria, it was not statistically meaningful to count the gold particles there.
The above experiment was repeated five times. The difference was examined using the post-hoc test (Fisher's protected least significant difference; Fisher's PLSD). The difference among pH 10.0, pH 7.0, and pH 2.0 in the most effective solution was statistically compared by the MannWhitney test.
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Results |
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In the control sections without heating, only a few gold particles showing positivity for the antibody were detected in the cytoplasm, some granules, the flagella, and the cell membrane. The antibody-positive granules contained several dense materials in which gold particles were distributed sporadically (Fig 1 Fig 2 Fig 3). In the sections without the primary antibody treatment, no gold particles were found. Gold particles in extracellular spaces were rarely observed.
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Under all heating conditions, better AR effects were generally observed in EDTA and Tris solution. During heating with the autoclave at 121C for 15 min, some grids floated to the surface of some fluids although some holes had been made in the lid to prevent floating by the imbalance of pressure. The sections that floated showed poorer staining than the sunken ones.
The control section without any AR treatment showed 3.3 ± 2.2 particles/granule (p/g).
At 121C for 15 Min (Table 1; Fig 1)
The positivity (5.7 ± 4.5 p/g) in Tris was stronger than control (p<0.0001). The stainability on flagella seemed to be better in EDTA, Tris, and S1699 than the control. The section in PB was less positive than control.
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At 99C for 40 Min (Table 1; Fig 2)
The positivity in EDTA, Tris, CA, or S1699 was stronger than the control (p<0.0001). The stainability on flagella was better in EDTA, Tris, urea, CA, and S1699 than the control. In granules, AR by Tris showed the best stainability.
At 65C for 24 Hr (Table 1; Fig 3)
In granules, except for PB, the positivity after AR was stronger than the control (p<0.0001). Except for DW and PB, the stainability on flagella was better in each solution than the control. The best staining on flagella was observed with AR by Tris. This heating condition was the most suitable for AR. Under this retrieval condition using Tris, the effect was examined at pH 10.0, 7.0, and 2.0 (Fig 4). AR at pH 10.0 showed the best effect (p<0.0001).
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Under all conditions, the stainability after using PB was weaker than that of the control. At 5% of significance level, antigen retrievals at 65C in Tris, 65C in EDTA, and 99C in Tris were more effective than at 99C in EDTA and 99C in S1699, which showed better stainability than those at 65C in DW, 65C in S1699, 65C in CA, 65C in urea, 99C in CA, and 121C in Tris. The order of efficiency of the antigen retrieval method could be surmmarized as follows: Tris (65C and 99C) and EDTA (65C) > EDTA (99C) and S1699 (99C) > DW (65C and 121C), S1699 (65C and 121C), CA (65C, 99C and 121C), urea (65C and 121C), EDTA (121C) and Tris (121C) > PB (65C, 99C and 121C) at 5% significance level. The difference among antigen retrievals at 65C in Tris, 65C in EDTA, and 99C in Tris could not be statistically determined.
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Discussion |
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In our study using the post-embedding method, the remarkable effects of AR by heating, as seen in light microscopy, were also found to be effective for immunoelectron microscopy. However, the stainability that we had expected of the immunoreaction was no better than that of the frozen sections. Staining of the samples after antigen protein had been viewed with skepticism in trials by light microscopy up to the present for fear of the induction of nonspecificity (
Various suitable temperatures for AR by light microscopy have been reported (
The effect of antigen retrieval was varied for each solution. The relationship between the AR solutions and morphological preservation was not examined in this study, but morphological preservation appeared to depend on the fixative used rather than on the AR solution. In trials by light microscopy, we have used various fluids for AR. With PB, the results with all heating experiments were poorer than the control. When we tried AR with Tris using an anti-urease antibody, we failed to achieve a better result than the control (not shown).
The mechanism of action of the AR solutions is still obscure. The optimal solution might also be dependent on the antibody used. Although a good effect produced in AR was believed to be manifested under a high pH of more than 8 or a low pH of less than 3 (
Antigen retrieval by heating of the section has been generally attributed to the breakage of formaldehyde-induced crosslinks between epitopes and extraction of diffusible blocking proteins, allowing better penetration of the antibody and increasing the accessibility of epitopes for the antibody to react (
The major antigen fraction detected by the antibody used in this study is the heat-stable O-antigen (lipopolysaccharide; LPS) (
For a start, ultrathin sections should be treated at 65C overnight in Tris to get better immunoprecipitation by immunoelectron microscopy using the post-embedding method. In conclusion, antigen retrieval by heating was effective at both the electron microscopic and the light microscopic level.
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Acknowledgments |
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We thank Dr Subrina Jesmin (Department of Cardiology Section, Hokkaido University Graduate School of Medicine) for the worthwhile discussions, Dr Eriko Shoji and Dr Yumiko Ohkochi for helpful assistance, and Prof Dr Hong Kean Ooi (Department of Veterinary Medicine, National Chung Hsing University, Taiwan) and Dr Yoichi Tari (DAKO Japan, Kyoto) for helpful advice.
Received for publication November 15, 2002; accepted March 21, 2003.
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Literature Cited |
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Andersen LP, Holck S, Povlsen O (1988) Campylobacter pylori detected by indirect immunohistochemical technique. Acta Pathol Microbiol Immunol Scand 96:559-564
Cattoretti G, Becker MHG, Key G, Duchrow M, Schlter C, Galle J, Gerdes J (1992) Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 168:357-363[Medline]
Cohen RJ, Fischer G, McNeal JE (2001) Immunostains after glutaraldehyde-based tissue fixation. Hum Pathol 32:242-243[Medline]
Isaacson PG (1994) Gastrointestinal lymphoma. Hum Pathol 25:1020-1029[Medline]
Iwamura M, Abrahamsson PA, Benning CM, Cockett ATK, di Santagnese PA (1994) Androgen receptor immunostaining and its tissue distribution in formalin-fixed, paraffin-embedded sections after microwave treatment. J Histochem Cytochem 42:783-788
Marshall BJ, Warren JR (1984) Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet I: 13111314
Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK (1991) Helicobacter pylori infection and the risk of gastric carcinoma. N Eng J Med 325:1127-1131[Abstract]
Rangell LK, Keller GA (2000) Application of microwave technology to the processing and immunolabeling of plastic-embedded and cryosections. J Histochem Cytochem 48:1153-1159
Ruizeveld de Winter JA, Trapman J, Vermey M, Mulder E, Zegers ND, van der Kwast TH (1991) Androgen receptor expression in human tissues: an immunohistochemical study. J Histochem Cytochem 39:927-936[Abstract]
Saito N (1990) Correlation between intracellular and extracellular lysozyme in acute (myelo)monocytic leukemia. Leukemia Lymphoma 2:347-350
Saito N, Konishi K, Sato F, Kato M, Takeda H, Sugiyama T, Asaka M (2003) Plural transformation-processes from spiral to coccoid Helicobacter pylori and its viability. J Infect 46:49-55[Medline]
Saito N, Pulford KAF, BretonGorius J, Mass JM, Mason DY, Cramer EM (1991) Ultrastructural localization of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes. Am J Pathol 139:1053-1059[Abstract]
Saito N, Sato F, Kato M, Takeda H, Sugiyama T, Asaka M (1998) Detection of coccoid Helicobacter pylori: light microscopical immunogold silver enhancing stain. Helicobacter 3:170-173[Medline]
Saito N, Takemori N, Hirai K, Kubota N, Onodera R, Namiki M (1992) Application of immunocryoultramicrotomy to free cells. Am J Hematol 39:223-225[Medline]
Sherburne R, Taylor DE (1995) Helicobacter pylori expresses a complex surface carbohydrate, Lewis X. Infect Immun 63:4564-4568[Abstract]
Shi SR, Cote RJ, Taylor CR (1997) Antigen retrieval immunohistochemistry: past, present and future. J Histochem Cytochem 45:327-343
Shi SR, Cote RJ, Taylor CR (2001) Antigen retrieval techniques: current perspectives. J Histochem Cytochem 49:931-937
Shi SR, Imam SA, Young L, Cote RJ, Taylor CR (1995) Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 43:193-201
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741-748[Abstract]
Stirling JW, Graff PS (1995) Antigen unmasking for immunoelectron microscopy: labeling is improved by treating with sodium ethoxide or sodium metaperiodate, then heating on retrieval medium. J Histochem Cytochem 43:115-123
Suurmeijer AJH, Boon ME (1993) Optimizing keratin and vimentin retrieval in formalin-fixed, paraffin-embedded tissue with the use of heat and metal salts. Appl Immunohistochem 1:143-148
Taylor CR, Shi SR, Cote RJ (1996) Antigen retrieval for immunohistochemistry. Status and need for greater standardization. Appl Immunohistochem 4:144-166
van den Berg FM, Baas IO, Polak MM, Offerhaus GJA (1993) Detection of p53 overexpression in routinely paraffin-embedded tissue of human carcinomas using a novel target unmasking fluid. Am J Pathol 142:381-385[Abstract]
Wilson DF, Jiang DJ, Pierce AM, Wiebkin OW (1996) Antigen retrieval for electron microscopy using a microwave technique for epithelial and basal lamina antigens. Appl Immunohistochem 4:66-71
Yi H, Leunissen JLM, Shi G, Gutekunst CA, Hersch SM (2001) A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level. J Histochem Cytochem 49:279-283