ARTICLE |
Correspondence to: S. Craig Cary, Graduate Coll. of Marine Studies, 700 Pilottown Rd, Lewes, DE 19958.
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Summary |
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New plastic resins are gradually replacing traditional paraffin-embedding in situ hybridization (ISH) strategies. One unique resin that has not been fully investigated or exploited with respect to light microscopic ISH is a methacrylate mixture. The advantage of this resin is its ability to be removed from tissues postsectioning, dramatically increasing hybridization signal compared to that obtained in other plastics. The goal of this study was to evaluate the general applicability of the methacrylate embedding acetone de-embedding (MEADE) technique for ISH investigations. Several high-resolution, high-sensitivity ISH protocols are described, using both end-labeled oligonucleotides and randomly primed DNA probes (200400 BPs), signal amplification by catalyzed reporter deposition (CARD), and chromogenic and fluorescent detection methods. With slight modification, the MEADE ISH technique permitted localization of bacterial symbionts in fragile gill tissue and collagen transcripts in foot tissue of two marine bivalves. MEADE ISH has proved extremely versatile and will likely be suitable for many future applications. (J Histochem Cytochem 46:149155, 1998)
Key Words: ISH (in situ hybridization), plastic resins, MEADE (methacrylate, embedding acetone, de-embedding), CARD (catalyzed reporter, deposition)
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Introduction |
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In situ hybridization (ISH) is now applied to address questions across a number of medical and scientific fields. With the technique's expansion, a variety of procedural modifications have developed that increase resolution and detection sensitivity of the hybridization. One such modification is the use of plastic resins as embedding media. These semithin plastic sections (12 µm) offer much greater preservation of cytological detail than traditional 47 µm-thick paraffin sections (
Methacrylates have been used in the past as embedding media for immunolabeling and ISH studies. Although sensitivity of RNA detection was reduced (
Our specific goals in this study were to extensively examine the utility of the MEADE technique in association with light microscopic ISH. Our research group required a plastic embedding protocol that would provide high morphological integrity and allow localization of low copy transcripts in marine invertebrate larvae. MEADE ISH was first applied to detect bacterial endosymbionts in the gills of the protobranch bivalve Solemya reidi. Although a previous ISH study localized dense concentrations of these symbionts in paraffin sections of adult gill filaments, the resolution and sensitivity of ISH were not sufficient to localize low-copy symbionts in poorly preserved paraffin-embedded larval tissues. MEADE ISH was applied in a second system to localize byssal thread collagen transcripts in the common blue mussel, Mytilus edulis. Future detection of transcripts for the collagen precursor preCol-P in M. edulis larvae also requires a sensitive ISH protocol with high resolution.
Both DIG- and biotin-labeled oligonucleotide probes and DIG-labeled double-stranded, randomly primed DNA probes were tested with MEADE ISH. Indirect chromogenic and fluorescent methods, as well as signal amplification by catalyzed reporter deposition (CARD) with the Renaissance Tyramide Signal Amplification (TSA) Kit, were successfully applied. The results have demonstrated the broad applicability of the MEADE technique and will enhance future ISH studies with methacrylate resins.
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Materials and Methods |
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Tissue Preparation
S. reidi gill filaments and M. edulis foot tissue were fixed overnight in 4% paraformaldehyde (pH 7.6), 0.5 M NaCl, 1 x MOPS (pH 7.0) at 8C. Tissues were washed in 1 x MOPS/0.5 M NaCl, aldehyde-quenched in 0.15 mM NH4Cl in wash buffer, and dehydrated through a graded ethanol series (50100%). From 100% EtOH, samples were transferred through an ethanolmethacrylate series (2:1, 1:1, 1:2 at -20C) before two or three changes in 100% methacrylate [80% butyl-methacrylate, 20% methyl-methacrylate, 0.5% (w/v) benzoin ethyl ether, and 10 mM DTT] and overnight storage (
Polymerization occurred in gelatin capsules 15 cm from the UV light source (two bulbs, 6 W each) at -8C for 12 hr. Block brittleness was reduced by placing six sheets of paraffin between a UV light and the samples. Wet sections (1.52 µm thick) were transferred onto 10-well hydrophobically coated glass slides with bioadhesive surface treatment (Erie Scientific; Portsmouth, NH), flattened with chloroform vapors, and dried.
Probes
S. reidi Probes.
Oligonucleotide probes were labeled with either DIG-ddUTP or biotin. The bacterial specific probe 338R (GCT GCC TCC CGT AGG AGT;
M. edulis Probes. Template for preCol-P probes was prepared by PCR amplification of cDNA inserts from preCol-P clones. A nonhomologous probe was prepared by similar amplification of the plasmid vector pBluescript (Stratagene; Palo Alto, CA). Double-stranded, DIG-dUTP-labeled DNA probes were synthesized by random priming using a Genius System Nonradioactive DNA Labeling and Detection Kit (Boehringer Mannheim). Efficiency of labeling was determined by dot-blot hybridizations. Average length of random primed probes was 200400 BPs.
MEADE In Situ Hybridizations
Although both S. reidi and M. edulis tissues were prepared using MEADE techniques, two hybridization protocols are described below because of different optimization requirements. All hybridizations were performed on 2050 sections per probe treatment, with sections taken from three to five different embedded block faces. Results were examined with a Nikon Microphot FXA epifluorescent microscope fitted with a dual bandpass filter set specific for visualization of fluorescein and propidium iodide.
S. reidi. A 10-min incubation in fresh 100% acetone removed the methacrylate resin from embedded tissue sections. Slides were washed in NaPBTw (20 mM Na2HPO4, 20 mM NaH2PO4, 0.9% NaCl, 0.1% Tween 20) (5 min), acetylated in 0.1 M triethanolamine (pH 8.0) with 0.5% acetic anhydride (3 min), washed, and prehybridized in hybridization buffer (HB1) (2 x SSC, 5 mM EDTA, 1 x Denhardt's solution, 0.5 mg/ml sheared herring sperm DNA, 0.1% Tween 20) (20 min). Hybridization occurred after addition of 1 ng/µl labeled probe in HB for 712 hours at 46C. Sections were washed (15 min each) in 2 x SSC, 0.1% Tween 20 at 46C and 0.2 x SSC, 0.1% Tween 20 cooling to room temperature (RT). Hybrid detection was performed according to one of the following strategies.
Strategy 1: NBT/BCIP Chromogenic Detection of DIG. Sections were blocked (15 min at RT) with 2 mg/ml BSA in NaPBTw, incubated with alkaline phosphatase (AP)-conjugated anti-DIG antibody (Boehringer Mannheim; 1:2000 in blocking buffer with 10% sheep serum at 37C; 3 hr), and washed in NaPBTw (5 min) and AP buffer (100 mM Tris-HCl, pH 9.6, 100 mM NaCl, 50 mM MgCl2; 2 min). The AP-conjugated antibody was detected with 4.5 µl/ml nitroblue tetrazolium (NBT)/3.5 µl/ml 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Boehringer Mannheim) and 1 x levamisol (Vector Laboratories; Burlingame, CA) in AP buffer for 12 hr. Sections were rinsed, counter-stained with pyronin YMethyl green, and examined.
Strategy 2: CARD Amplification/ Fluorescent Detection of Biotin. Endogenous biotin was blocked with the Avidin/Biotin Blocking Kit (Vector Laboratories) after tissue acetylation and before hybridization. For hybrid detection, protocols for the TSA-Indirect Kit (NEN Life Science Products; Boston, MA) were followed according to the manufacturer's instructions. After application of streptavidinhorseradish peroxidase (HRP), sections were incubated with biotinyl tyramide (8 min) and then streptavidinfluorescein (FITC). Propidium iodide served as counterstain (Molecular Probes; Eugene, OR).
M. edulis Optimization. Optimal length of acetone treatment was investigated by incubating tissues in 100% acetone for 018 minutes and comparing hybridization signal using detection Strategy 3 for chromogenic development (described below). Sections were also subjected to proteolytic digestion with proteinase K (Sigma Chemical; St Louis, MO) (0.2, 2, 10, and 20 µg/ml at 37C), pepsin (Sigma) (0.2 and 2 µg/ml), or dilute hydrochloric acid (0.2 M) solutions before hybridization for ultrastructural/signal comparison.
M. edulis. Slides were de-embedded in acetone (1215 min), washed in NaPBTw, and incubated in 10 µg/ml proteinase K (4 min, 37C). The reaction was stopped by incubation in 2% glycine in NaPBTw, and then sections were acetylated, washed, and prehybridized in HB2 (50% formamide, 4 x SSC, 1 x Denhardt's solution, 0.2 mg/ml sheared herring sperm DNA, 5% dextran sulfate). Dilutions of 1:500 DIG-labeled preCol-P probe or nonhomologous probe were used for overnight hybridizations (42C). Tissues were washed in 2 x SSC and 0.1 x SSC at 42C. When possible, all solutions were prepared with 0.1% diethyl pyrocarbonate-treated water (DEPC; Sigma). Blocking and detection of the DIG hybrid complex are described below.
Strategy 3: Vector Blue Chromogenic Detection of DIG. Sections were preblocked with 2% blocking buffer (Boehringer Mannheim) and incubated with AP conjugated anti-DIG Fab fragments (Boehringer Mannheim; 1:200 for 1.53 hr at 37C). After washes in 100 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20, and development buffer [10 mM Tris-HCl (pH 8.2)], hybrids were detected with Vector Blue Substrate Kit reagents (Vector Laboratories; 20 min). Sections were counterstained with 1% Neutral Fast Red (Sigma) and viewed.
Strategy 4: CARD Amplification/ Sandwiched Vector Blue Chromogenic Detection of DIG. Tissues were preblocked with the Avidin/Biotin Blocking Kit and then 2% blocking buffer. After detection of probe-target hybrids with biotin-SP-conjugated anti-digoxin (Jackson ImmunoResearch Laboratories; West Grove, PA) (1:200 for 1.53 hr at 37C), TSA-Indirect Kit protocols were followed for application of streptavidinHRP and biotinyltyramide (6 min). Tissues were washed, incubated with an AP-conjugated biotinavidin complex (Vectastain ABC-AP Kit; Vector Laboratories), and developed chromogenically with Vector Blue Substrate.
Strategy 5: CARD Amplification/ Fluorescent Detection of DIG. Methods were followed as above in Strategy 4. However, after the application of biotinyltyramide and TSA washes, hybrids were detected with streptavidinFITC and counterstained with propidium iodide (Molecular Probes).
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Results |
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To test the general applicability of MEADE in conjunction with ISH, a variety of probing and detection strategies were performed. To facilitate comparisons among these five detection strategies, the major steps of each are outlined in Figure 1.
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The MEADE technique provided excellent resolution and integrity of cellular morphology in semithin sections of fragile S. reidi gill filaments. For structural orientation, tightly packed ribbon bands represent the gill lamellae of a single gill filament. The lamella is approximately two cells in width and is composed only of vascular tissue and an outer layer composed of bacteriocytes (containing symbionts) and thin intercalary cells that lack symbionts. Higher magnifications of the gill lamellae show large host cell nuclei surrounded on both sides by smaller, granule-like symbiotic bacteria (Figure 2).
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Hybridization results using each detection strategy are shown in Figure 3 and Figure 4. The 16S rRNA of bacterial endosymbionts of S. reidi was successfully localized within host bacteriocytes along the proximal portions of the gill lamellae (Figure 3). Figure 3A and Figure 3B were developed using Strategy 1 for chromogenic detection of the hapten DIG. The NBT/BCIP reacts with the conjugated AP to produce a purple precipitate wherever the hybrid complex is localized within the tissue. The nonhomologous probe produced minimal background nonspecific labeling (Figure 3A), whereas strong positive labeling of bacterial symbionts was noted in tissues hybridized with the symbiont-specific probe SR-1254R (Figure 3B). Figure 3C and Figure 3D were each developed using CARD amplification and fluorescent detection (Strategy 2). Figure 3C shows hybridization results with the nonhomologous probe Mefp-3.5T. The propidium iodide counterstain (specific for nucleic acids) allows large host cell nuclei to be easily distinguished from surrounding granular bacterial symbionts. The green fluorescent precipitate in Figure 3D corresponds with positive labeling of the endosymbionts, after tissue hybridization with the symbiont-specific probe SR-1254R.
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Several modifications were required to adapt the original MEADE ISH procedure for M. edulis tissues and double-stranded, randomly-primed DNA probes (200400 BPs). Although preCol-P transcripts could be detected in all acetone-treated sections (018 min), maximal signal was observed in those de-embedded for 1215 min. Proteolytic digestion of tissue enhanced initial low hybridization signals. Typically, higher concentrations and/or longer tissue digestion resulted in both greater signal intensity and loss of tissue morphology. Digestion with proteinase K (10 µg/ml; 4 min at 37C) provided the best results, with minimal loss of morphology.
MEADE ISH reactions were conducted to localize transcripts of the byssal collagen precursor, preCol-P, in M. edulis foot tissue. Foot tissue is mostly glandular, with some striated muscle running throughout (Figure 4). Probe hybrids were localized in patches throughout most of the glandular tissue; the globe-shaped muscle tissue located in the right portion of each section did not contain any preCol-P transcripts and therefore served as an internal negative control (Figure 4AD). Figure 4A and Figure 4B were both developed chromogenically. Figure 4A was detected using Strategy 3; faint labeling of preCol-P transcripts was observed. In contrast, Figure 4B was detected using Strategy 4 with CARD amplification. Although not shown, nonhomologous probe controls produced negligible background staining in each hybridization. Hybridizations were conducted on M. edulis foot tissue and were developed using Strategy 5 for CARD amplification/fluorescent detection of DIG (Figure 4C and Figure 4D). Labeling in Figure 4D is similar to that shown in Figure 4B. Localization of transcripts for preCol-P using CARD amplification and chromogenic or fluorescent detection was substantially increased compared to chromogenic detection alone (Figure 4A).
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Discussion |
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This study was undertaken to evaluate the utility of the methacrylate embedding, acetone de-embedding (MEADE) technique for light microscopic ISH studies. This technique was examined in conjunction with various marine systems, hapten-labeled probes, and detection strategies. Despite documentation of superior cellular detail and high hybridization signal (
In addition to the methacrylate resin, two alternative plastic resins (LR White and Lowicryl K4M; Ted Pella, Tustin, CA) were initially examined to determine which provides maximal structural preservation and integrity of tissues. Neither LR White nor Lowicryl K4M allows tissue de-embedding before hybridization. The methacrylate resin permitted optimal retention of cell morphology and visualization of fine structural detail in semithin tissue sections of adult S. reidi gill filaments. It also provided maximal ISH labeling efficiency (results not shown). The reduced labeling of rRNA sequences found in LR White- and K4M-embedded filaments compared to the de-embedded methacrylate tissue was expected because of differences in probe accessibility through the embedding resins. Specifically, probe hybridization in LR White and Lowicryl K4M sections is probably restricted to those sites at the surface of the tissue section ("surface-only" labeling).
We developed the MEADE hybridization protocol using short digoxigenin (DIG) end-labeled oligonucleotide probes specific for the 16S rRNA of bacterial endosymbionts in the gill tissue of S. reidi (a single reporter molecule theoretically maximized probe penetrability, compared to the potential steric hinderance resulting from incorporation of multiple hapten molecules conjugated onto larger probes). A chromogenic method (Strategy 1) enabled successful hybrid detection, with strong hybridization signal and low levels of background (Figure 3A and Figure 3B). Once the initial ability to perform ISH with the methacrylate de-embedded tissue was confirmed, it became the goal of this study to examine the possible limitations of the technique by testing a variety of probes, tissues, and detection strategies.
The versatility of the MEADE ISH protocol was first examined with respect to fluorescence and the application of biotinylated oligonucleotide probes. The TSA-Indirect Kit relies on catalyzed reporter deposition (CARD) amplification of biotinyl tyramide. The newly immobilized biotin source is then detected by standard indirect methods using fluorescent steptavidin conjugates. The technique has proved extremely sensitive for in situ hybridizations and signal amplification of low target signal (
In our study, hybridization signals were strong and clearly distinguishable from low background staining and the propidium iodide counterstain (Figure 3C and Figure 3D). Although labeled regions corresponded to those identified with DIG detection and NBT/BCIP development, direct comparison between chromogenic and fluorescent detection systems was not possible because of the different qualities of each reaction.
MEADE in situ reactions were tested using a second marine system and longer DIG-labeled randomly primed, double-stranded DNA probes (average length 200400 BPs). A longer incubation time in acetone (1215 min) and a brief proteolytic digestion of the tissue were required to optimize intensity of hybridization signal with minimal loss of morphology. After optimization within the M. edulis system, various detection methods were investigated.
Strategy 1 (NBT/BCIP, Figure 3B) and Strategy 3 (Vector Blue, Figure 4A) development are directly comparable because both rely on an anti-DIG Fab fragment to bind to the DIG hapten. The antibody's conjugated AP molecule reacts chromogenically with the Vector Blue substrate and NBT/BCIP. However, in M. edulis, dissimilar counterstaining made the blue hybridization signal preferable to the purple NBT/BCIP. In addition, greater signal resolution and localized development were noted in this tissue with the Vector Blue substrate.
Both Strategy 4 and Strategy 5 utilized CARD signal amplification. Strategy 4 integrated catalyzed reporter deposition of biotinyl tyramide with the Vectastain ABC-AP Kit. This kit employs a preformed APbiotin/avidin macromolecular complex (the ABC-AP complex), which can then be chromogenically developed as in Strategy 3. Detection in Strategy 5 was directly comparable to Strategy 2 using the TSA Kit. Chromogenic and fluorescent detection produced similar results, each yielding substantial signal amplification (Figure 4B and Figure 4D). All detection strategies (Strategies 35) reproducibly localized the mRNA of the byssal collagen precursor in glandular foot tissue.
To conclude, the results of this study have shown the versatility of the MEADE technique in conjunction with light microscopic ISH. The methacrylate resin was ideal for fragile tissues that required high retention of cellular morphology, and the semithin (12 µm) sections reduced microscopic out-of-focus blur. Hybridizations were successfully completed in two different marine systems to localize mRNA and rRNA transcripts. Hybrids were detected with several different probe molecules (oligonucleotide and randomly primed), probe labels (biotin and DIG), signal amplification (CARD), and detection strategies (chromogenic and fluorescent). MEADE ISH will undoubtedly prove invaluable for future studies that aim to localize transcripts at the cellular and/or subcellular level throughout early embryological stages of development in other marine larvae. Moreover, we expect that advances in fluorescent sandwiching, cyanine fluorochromes (Cy5 and Cy7), and charge-coupled device (CCD) cameras will soon be applied to the MEADE ISH protocols for even more qualitative and quantitative studies.
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Acknowledgments |
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Supported by grants from the National Science Foundation (OCE-9016373 and OCE-9217950 to SCC) and the National Institute of Health (3-2-21-3501-06 to JHW).
We would like to thank the captain and crew of the R/V Alta (Bamfield Marine Research Station, British Columbia, Canada) for their perseverance in collecting S. reidi specimens for this study. NEN Life Science Products generously donated materials for our use as well as technical assistance. Kirk Czymmek provided a critical review of the manuscript and Monroe Givens was helpful with reproduction of digital images.
Received for publication April 16, 1997; accepted August 7, 1997.
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