ARTICLE |
Correspondence to: Stephanie Groos, Center of Anatomy, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail: groos.stephanie@mh-hannover.de
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Summary |
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Epoxy resins provide optimal tissue morphology at both the light and the electron microscopic level and therefore enable correlative studies on semithin and thin sections from the same tissue block. Here we report on an approach to retain these advantages for immunolabeling studies by adapting and combining well-known techniques, i.e., surface etching with sodium ethoxide and heat-mediated antigen retrieval. We propose a simple procedure for immunostaining semithin and thin epoxy resin sections. To check its applicability, well characterized, commercially available antibodies (against E-cadherin, -catenin, and
-catenin) were used on sections of human small intestine. By light microscopy, the immunostaining efficiency was compared on cryo-, paraffin, and epoxy semithin sections processed in parallel. The most detailed results were obtained on semithin sections, where the labeling precisely delineated the lateral plasma membrane of the enterocytes. At the electron microscopic level the procedure did not damage the structures and allowed an efficient, reproducible immunogold labeling extending homogeneously over exceptionally wide tissue areas. The three antibodies specifically labeled the zonula adherens of the junctional complex between epithelial cells and, in agreement with light microscopic observations, the lateral plasma membrane. (J Histochem Cytochem 49:397406, 2001)
Key Words:
E-cadherin, -catenin,
-catenin, adherens junction, etching, antigen retrieval, immunogold labeling
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Introduction |
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THE MAIN CRITERION for the choice of an immunohistochemical procedure is an efficient and reproducible labeling of the antigen together with preserved tissue morphology. However, if the correlative light (LM) and electron microscopic (EM) methods that are routinely used in morphological studies could be applied in analyzing immunostaining, this would be a valuable advantage. This can be achieved with biological specimens (cells and tissues) embedded either in modern acrylic media (Lowicryl, LR White, LR Gold) or in epoxy resins (Araldite, Epon) that warrant the best morphology. However, the literature shows that this possibility has seldom been taken into consideration (-catenin, and
-catenin on the human small intestinal epithelium. These adhesion molecules were chosen because of their essential role in cell turnover along the cryptvillous axis and because they have already been investigated in the small intestine of rodents using immunofluorescence (
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Materials and Methods |
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Tissue Preparation
Samples of human proximal jejunum were surgically removed from three patients (informed consent was obtained in accordance with the Helsinki Declaration of 1975) from the edges of the anastomosing sites during the preparation of a Roux-en-Y loop (
Primary Antibodies and Specificity
Monoclonal primary antibodies generated in mice (Transduction Laboratories; Lexington, KY) were used. They were directed against the C-terminal domains of E-cadherin (clone 36, catalogue no. C20820), -catenin (clone 5, catalogue no. C21620), and
-catenin (clone 14, catalogue no. C19220). The specificity of the antibodies was checked as recommended by the manufacturer by immunoblotting of lysates provided together with the antibodies: A 431 cells for E-cadherin, human endothelial cells for
-catenin, and HeLa cells for
-catenin. The polypeptides in the lysates were separated by SDS-PAGE using a 919% gradient polyacrylamide gel and electrophoretically transferred to a Protran nitrocellulose membrane (Schleicher & Schuell; Dassel, Germany). The membrane was blocked with 5% skimmed milk and incubated for 60 min with the primary antibodies diluted according to the instructions of the manufacturer. For detection, the blots were incubated for 60 min with peroxidase-conjugated goat anti-mouse IgG (Cappel; Organon Teknika, West Chester, PA), developed with the chemiluminescent reagent NEN (Nalgene; Boston, MA) and exposed on Hyperfilm (Amersham Life Science; Little Chalfont, UK). The antibodies recognized bands at 120 kD (anti-E-cadherin), 102 kD (anti-
-catenin), and 92 kD (anti-
-catenin) (Fig 1) in agreement with published data.
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Labeling Reagents
For immunohistochemistry, a biotin-conjugated affinity-purified goat anti-mouse IgG (Jackson ImmunoResearch Laboratories; West Grove, PA) was used as secondary antibody. For cryosections, a dichlorotriazinylfluorescein (DTAF)-labeled streptavidin (Jackson ImmunoResearch) was applied, while for both paraffin and epoxy resin sections peroxidase-labeled streptavidin (Jackson ImmunoResearch) was used. In thin sections, the immunoreaction was visualized with a goat anti-mouse IgG+IgM antibody conjugated to 10-nm gold particles (British BioCell; Cardiff, UK). All incubation steps were carried out in a humid chamber.
Immunohistochemical Procedures
Cryosections (8 µm thick) of frozen samples were cut with a cryostat Leica CM 1900 (Leica; Bensheim, Germany) at -20C, collected on SuperFrost Plus microscope slides (MenzelGläser; Braunschweig, Germany), air-dried, and rehydrated. Because formaldehyde-fixed tissue was used, in some cases antigen retrieval was performed by transferring the cryosections for 30 min into 0.01 M citrate buffer, pH 6.0, previously heated in a microwave oven to 95C.
After washing in PBS and blocking nonspecific protein binding with 5% BSA in PBS for 30 min at room temperature (RT), the sections were incubated overnight at 4C with the primary antibodies as indicated in Table 1. The sections were washed in PBS, treated with the biotin-labeled secondary antibody (5 µg/ml) for 60 min (RT) and then with streptavidin labeled with the fluorochrome DTAF (5 µg/ml) for 30 min at RT. After mounting with Mowiol (Sigma; Deisenhofen, Germany), the sections were examined with a Leitz Dialux 22 microscope equipped with fluorescence. Photomicrographs were taken on Kodak TMAX 400 film (Eastman Kodak; Rochester, NY).
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Paraffin sections (about 4 µm thick) were cut and collected on glass slides pretreated with 3-(triethoxysilyl)propylamine (silane; Merck, Darmstadt, Germany). The sections were dewaxed in xylene and rehydrated in descending concentrations of ethanol. Endogenous peroxidase activity was blocked by treating the sections with 0.6% H2O2 in 96% ethanol for 20 minutes at RT. Later, these sections were processed for antigen retrieval and immunostaining in the same way as the epoxy resin semithin sections (see below).
For epoxy resin sections, semithin sections (about 1 µm thick) were cut with a Leica Ultracut R ultramicrotome, collected on silane-treated slides, and air-dried at RT. A controlled corrosion of the section surface was obtained by etching the sections for at least 15 min (shorter etching times were insufficient) in a solution composed of saturated sodium hydroxide in absolute ethanol (sodium ethoxide) diluted to 50% with absolute ethanol and subsequently rehydrating in descending concentrations of ethanol. After rehydration, for both paraffin and epoxy sections antigen retrieval was performed in a microwave oven (Sharp R-2S67; Sharp, Hamburg, Germany) by heating the sections in 0.01 M citrate buffer, pH 6.0 (
Nonspecific protein binding was blocked by treating the sections with 5% BSA dissolved in PBS for 30 min at RT. After a short rinse with PBS, the sections were incubated overnight at 4C with the primary antibodies diluted as indicated in Table 1, washed in PBS, treated for 60 min with biotinylated goat anti-mouse IgG (5 µg/ml), and then with peroxidase-labeled streptavidin (5 µg/ml) for 30 min, both at RT. Peroxidase labeling was visualized with 3,3'-diaminobenzidine (Sigma) as chromogen. The sections were washed in PBS and subsequently in distilled water, dehydrated in ascending concentrations of ethanol, cleared in xylene, mounted in Entellan (Merck, Darmstadt, Germany) and examined with a Leica DMLB microscope.
Thin sections (about 70 nm thick) were prepared from the same block used for LM immunohistochemistry and collected on nickel grids. During the immunostaining procedure, incubation steps were carried out by floating the sections on 20-µl drops of each solution; washing steps were performed by dipping the grids into the washing solution. Epoxy resin was partially corroded by etching the sections for 10 sec in saturated sodium ethoxide diluted to 50% with absolute ethanol. Etching times of 5, 10, 15, 20, and 25 seconds had been tested in advance (see Results). After rehydration and washing in distilled water, antigen retrieval was carried out by heating the sections for 10 min to 95C and then cooling them to 21C at a rate of 0.04C/sec in a thermocycler UNO-Thermoblock (Biometra; Göttingen, Germany). In preliminary tests, times of 5, 10, and 15 min for antigen retrieval had been checked. After washing in three changes of PBS, nonspecific protein binding was blocked by incubation with 5% BSA in PBS for 30 min at RT. Incubation with the various primary antibodies (for working concentrations see Table 1) was carried out overnight at 4C. After application of the 10-nm gold-labeled secondary antibody for 60 min at RT at a concentration of 0.8 µg/ml and subsequent washing in PBS, the immunoreaction was stabilized with 2.5% glutaraldehyde in PBS for 10 min (
Control Experiments
Control sections were treated in the same way as the experimental sections, but either omitting the primary antibodies or replacing them with normal mouse serum in the same concentrations as the applied antibodies.
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Results |
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Light Microscopy
After application of antibodies against E-cadherin, low magnification of cryo-, paraffin, and epoxy sections showed immunostaining of the epithelium (Fig 2a2c). The staining was diffuse in cryosections, but in paraffin and epoxy sections it was localized to the lateral plasma membrane of the enterocytes throughout the cryptvillous axis. Around the goblet cells the staining was more pronounced (Fig 2a2c).
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Although cryosections did not require pretreatment for detection of E-cadherin and catenins, their immersion in citrate buffer previously heated in a microwave oven improved the pattern of the fluorescent staining, which appeared more confined to the plasma membranes and in places had a dotted appearance (Fig 2a and Fig 2d).
To achieve homogeneous and reliable staining on paraffin sections, heating in a microwave oven was necessary. Epoxy sections were not labeled without a sequential pretreatment of surface corrosion (etching) with sodium ethoxide and microwave heating. In both paraffin (Fig 2b and Fig 2e) and epoxy sections (Fig 2c and Fig 2f) the immunolabeling was always restricted to the plasma membrane of the epithelial cells and had a dotted appearance. This aspect was particularly evident in epoxy sections (Fig 2f and Fig 3a). In cryosections, the very intense fluorescence at the crypt base prevented clear immunostaining of single cells (Fig 2a). In contrast, in paraffin (Fig 2b) and even more so in epoxy sections (Fig 2c and Fig 3a), the Paneth cells were morphologically recognizable because of their granules and a pronounced specific labeling of the lateral plasma membrane, whereas the basal plasma membrane remained unlabeled (Fig 2b, Fig 2c and Fig 3a). This peculiar staining of Paneth cells was especially evident with Nomarski interference optics (Fig 3a) and was confirmed for all three antibodies applied (data not shown).
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Electron Microscopy
To obtain optimal labeling without loss of specificity, it was necessary to evaluate etching time, different concentrations of the etching reagent, and various heating periods for antigen retrieval. Thin sections that were etched for 5 sec in sodium ethoxide diluted to 50% or for 20 sec in a dilution of 25% and subsequently heated showed good morphological preservation but only poor labeling. In contrast, if the sections were subjected to 15, 20, or 25 sec of etching in 50% solution, labeling improved but the long etching periods caused serious damage to the sections, such as the formation of holes, the size of which increased during heating. In extreme cases the sections were reduced to small remnants close to the bars of the grid. Concerning the heating time a threshold of 10 min was set, the labeling being insufficient below this time and not enhanced by prolonged exposure. Therefore, in our approach the best and most reliable results were obtained by etching the thin sections for 10 sec in 50% sodium ethoxide and subsequently heating them to 95C for 10 min, followed by a gradual, controlled decrease of temperature to 21C (see Materials and Methods). This procedure did not damage the morphology and allowed analysis at the EM level of the immunolabeling throughout the whole section over an area equivalent to that of a semithin section, as shown in Fig 3a (LM) and 3b (EM) at the same magnification. A survey of cells and/or tissue areas could be obtained and specific structures could be examined by high magnification using the same (Fig 3c and Fig 3d) or adjacent sections (Fig 4e). Alternatively, immunolabeling first detected at high magnification (Fig 3d) could be traced back via intermediate (Fig 3c) to survey magnification (Fig 3b) to establish the exact position of the labeled epithelial cells along the cryptvillous axis.
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In human small intestine, all three antibodies showed immunoreactions at the level of the junctional complex between enterocytes, where the gold particles preferentially bordered the cytoplasmic sides of the zonula adherens but left free both zonula occludens and desmosomes (Fig 4a and Fig 4c). However, some particles appeared dislocated inside the intercellular space (Fig 4a and Fig 4c). A bilateral correspondence of the gold labeling could occasionally be observed between two enterocyte plasma membranes (Fig 4b). Immunostaining was always pronounced at the junctions between enterocytes and goblet cells (Fig 4d) and especially between two adjacent Paneth cells (Fig 4e). Furthermore, in agreement with LM, the immunolabeling was localized with varying density below the junctional complex along the lateral plasma membrane of all epithelial cells (villus and crypt; Fig 4d and Fig 4f). Reliable and specific labeling was obtained with all three antibodies. However, the lowest density of labeling was seen with the antibody against E-cadherin (compare Fig 4a with Fig 4b and Fig 4c).
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Discussion |
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Advantages Over Other Procedures of Epoxy Sections for Immunostaining
Once a method to remove the embedding medium from epoxy semithin sections was found (
Acrylic resins such as Lowicryl, LR White, and LR Gold are preferable for several reasons. They are water-compatible and therefore require only a partial dehydration of the tissue, thus improving its reactivity with antibodies. Second, the hydrophilic properties of the plastic medium reduce background staining and make these resins especially suited for postembedding immunocytochemistry (
LR White and LR Gold might have other technical drawbacks, such as tissue extraction and shrinkage caused by its exposure to the damaging influence of the plastic monomer during the embedding procedure, depending on the temperature conditions under which polymerization occurred (
Cryo-ultrathin sections, already used for enzyme histochemistry in the 1960s (
Antigen retrieval is a section pretreatment that exposes hidden epitopes (
Localization of E-cadherin and Catenins
E-cadherin, -catenin, and
-catenin belong to a family of adhesion molecules that are expressed at the junctions of the adherens type (for review see
In agreement with previous investigations (-catenin, and
-catenin are localized along the lateral plasma membrane of the enterocytes. As shown by others (
- and
-catenin. In our study they provide the best morphological tissue preservation, together with efficient and reliable immunostaining. Semithin sections can be prepared that allow a survey of the spatial distribution of the antigen with the option to study its subcellular expression on consecutive thin sections.
In thin epoxy sections of human small intestine, E-cadherin and/or its associated proteins - and
-catenin are distinctly localized at the zonula adherens. The tight junction zone and the desmosomes are free of immunogold particles. Our findings are in agreement with electron microscopic investigations of different segments of animal and human intestine, using either acrylic resin sections (
The results of this study demonstrate that epoxy sections from the same tissue block are suitable for performing immunostaining at both the light and electron microscopic level by maintaining all advantages these media have: best tissue preservation, good infiltration qualities, block samples easy to cut, excellent structure contrast, and stability under the electron beam.
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Acknowledgments |
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We wish to thank Prof Dr G. Hünefeld (GöttingenWeende) for help in collecting the specimens, Prof Dr E. Ungewickell for critical reading of the manuscript, Ms C. Lichtenberg for technical assistance, Ms. A. Hundt for photographic assistance, and Ms S. Fryk for linguistic correction of the manuscript.
Received for publication June 21, 2000; accepted October 13, 2000.
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