PLs = postlarvae; LM = light microscopy; EM = electron microscopy; PCR = polymerase chain reaction.
| 1. | Standard Screening Methods for WSSV
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| | 1.1. | Nested polymerase chain reaction of tissues and haemolymph
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| | | The protocol described here is according to Lo et al. (28, 32), although alternative assays have been described (21, 22, 24, 46, 53). In brief, collect living shrimp specimens and, using a disposable stick, homogenise approximately 100-200 mg of tissue (or use 100 µl of haemolymph) from live juvenile to subadult shrimp, postlarvae 11 upwards (PL11 up) with removed heads, or whole PL10 downwards in a 1.5 ml microfuge tube with 600 µl of lysis solution (100 mM NaCl, 10 mM Tris/HCl, pH 8, 25 mM EDTA [ethylene diamine tetra-acetic acid], 0.5% SLS [sodium N-laurylsarcosinate] or 2% SDS [sodium dodecyl sulfate], and 0.5 mg/ml proteinase K added just before use). Except for PL10 or below, be careful to exclude shrimp eyes from samples, as these are known to contain PCR inhibitor(s) (28, 32).
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| | | After homogenisation, incubate at 65°C for 1 hour. (If the virus load is very low, or if very high quality, less degraded genomic DNA is required, start with 1000 µl of lysis solution and insert this step: centrifuge at 13,000 g for 5 minutes and transfer 600 µl of the supernatant to a fresh 1.5 ml tube.) Add 5 M NaCl to a final concentration of 0.7 M. Next slowly add 1/10 volume of N-cetyl N,N,N-trimethylammonium bromide (CTAB)/NaCl solution (10% CTAB in 0.7 M NaCl) and mix thoroughly. Incubate at 65°C for 10 minutes, and then, at room temperature, add an equal volume of chlorofoum/isoamyl alcohol (24/1) and mix gently. Centrifuge at 13,000 g for 5 minutes and then transfer the aqueous solution (upper layer) to a fresh 1.5 ml tube and add an equal volume of phenol. Mix gently and centrifuge at 13,000 g for 5 minutes.
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| | | Collect the upper layer solution and repeat the phenol extraction process 1 to 2 times. Transfer the final upper layer to a new tube, mix gently with two volumes of chloroform/isoamyl alcohol (24/1) and centrifuge at 13,000 g for 5 minutes. Transfer the upper layer to a new tube and precipitate DNA by adding two volumes of 95% or absolute ethanol followed by standing at -20°C for 30 minutes or -80°C for 15 minutes. Centrifuge at 13,000 g for 30 minutes and discard the ethanol. Wash the DNA pellet with 70% ethanol, dry and resuspend in 100 µl sterilised double-distilled water at 65°C for 15 minutes. Use 1 µl of this DNA solution for one PCR reaction.
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| | | For the first-step PCR reaction, add 1 µl DNA template solution (containing about 0.1-0.3 µg DNA) to a PCR tube containing 100 µl of reaction mixture (10 mM Tris/HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200 µM of each dNTP, 100 pmol of each primer, 2 units of thermal stable DNA polymerase). The outer primer sequences are 146F1, 5'-ACT-ACT-AAC-TTC-AGC-CTA-TCT-AG-3' and 146R1, 5'-TAA-TGC-GGG-TGT-AAT-GTT-CTT-ACG-A-3'. The PCR profile is one cycle of 94°C for 4 minutes, 55°C for 1 minute, and 72°C for 2 minutes, followed by 39 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes and a final 5-minute extension at 72°C. The WSSV-specific amplicon from this reaction is 1447 bp. The sensitivity is approximately 20,000 copies of a plasmid template.
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| | | For the second-step of the (nested) PCR reaction, add 10 µl of the first-step PCR reaction product to 90 µl of PCR cocktail of the same composition as above except that it contains the second (inner) primer pair: 146F2 (5'-GTA-ACT-GCC-CCT-TCC-ATC-TCC-A-3') and 146R2 (5'-TAC-GGC-AGC -TGC-TGC-ACC-TTG-T-3'). Use the same PCR amplification protocol as above. The WSSV-specific amplicon from this reaction is 941 bp. The overall sensitivity of both steps is approximately 20 copies of a WSSV plasmid template.
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| | | To visualise, electrophorese 10 µl of PCR reaction products on 1% agarose gels containing ethidium bromide at a concentration of 0.5 µg/ml. Decapod-specific primers (143F 5'-TGC-CTT-ATC-AGC-TNT-CGA-TTG-TAG-3' and 145R 5'-TTC-AGN-TTT-GCA-ACC-ATA-CTT CCC-3' yielding an 848 bp amplicon; N represents G, A, T, or C) are available to verify the quality of the extracted DNA (32) and the integrity of the PCR reaction. A positive control (WSSV DNA template) and negative controls (no template and shrimp DNA template) should be included in every assay.
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| | 1.2. | PCR of postlarvae
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| | | From a nursery or hatchery tank containing 100,000 postlarvae (PL) or more, sample approximately 1000 PL from each of five different points. Pool the samples in a basin, gently swirl the water in the basin and then select an assay sample from living PL that collect at the centre of the basin. Choose the sample number according to the assumed prevalence (see Chapter I.3.). For PL11 and above be careful to exclude shrimp eyes from samples, as these are known to contain PCR inhibitor(s) (28, 32). Homogenise the sample in an appropriate volume of lysis solution (see Section 1.1.) and then follow the procedure for nested PCR with a 600 µl fraction as described in Section 1.1. Homogenates that have been stored in lysis solution at room temperature for more than 1 year can still be used for PCR, but prolonged storage in lysis solution decreases the DNA quality and reduces assay sensitivity.
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| | 1.3. | Antibody-based assays
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| | | Both polyclonal (PAbs) and monoclonal antibodies (MAbs) to the virus have been used for the detection of WSSV in clinical specimens.
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| | | The protocol described here for the production and application of polyclonal antibodies for WSSV detection is from Nadala et al. (44) and Loh et al. (34). This includes purification of WSSV virions from laboratory-infected shrimp, generation of immunoglobulins (Ig) in New Zealand white rabbits, purification of the IgG using recombinant bacterial protein-G columns, and removal of cross-reacting normal shrimp antigens by adsorption on to acetone-dried, ground shrimp muscle tissue and haemolymph. For assay, remove 0.1 ml of haemolymph from live shrimp specimens and dilute in an equal volume of citrate buffer (19.3 mM sodium citrate, 239.8 mM NaCl, 182.5 mM glucose, 6.2 mM EDTA, pH 7.0) for immediate use or storage at -80°C until used. For Western blotting, use a 200 µl sample, clarify at 8000 g for 5 minutes and then pellet the supernatant at 140,000 g for 5 minutes. Resuspend pellets in 100 µl 2 x loading buffer (2.5 ml 0.5 mM Tris/HCl, pH 6.8, 4 ml 10% SDS, 2 ml glycerol, 1 ml beta-mercaptoethanol, and 0.5 ml deionised distilled water) and heat at 95°C for 5 minutes. Load a 10 µl sub-sample on to 10% SDS-PAGE (polyacrylamide gel electrophoresis), and electrophorese at 200 V. Blot the electrophoresed gel on to a nitrocellulose membrane (0.1 µm pore size) in blotting buffer (3.03 g Tris base, 14.4 g glycine, and 200 ml methanol per litre) at 100 V for 1 hour. Rinse the membrane with phosphate buffered saline (PBS), pH 7.4, soak in 5% skim milk (in PBS) for 1 hour, and rinse with PBS for 5 minutes. Next, treat the membrane with a 1/1000 dilution of the primary antibody (IgG) for 1 hour, rinse three times with PBS for 5 minutes, and then treat with a 1/2500 dilution of goat anti-rabbit IgG-horseradish peroxidase conjugate for 1 hour. Rinse again three times with PBS for 5 minutes and then treat with the substrate, 3,3', 5,5'-tetramethylbenzidine (TMB), until a bluish purple colour develops. Stop the reaction by soaking the membrane in distilled water. All incubations should be carried out at 25°C±2°C. Use a purified viral preparation as a positive control and identify four major protein bands characteristic of WSSV at 19, 23.5, 27.5 and 75 kDa. The sensitivity is 1 ng of WSSV protein.
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| | | The protocol described here for the production and application of MAbs for WSSV detection is from Poulos et al. (49). Inject BALB/cByJ mice intraperitoneally with purified WSSV emulsified in synthetic adjuvant (MPL+TDM [monophosphoryl lipid A and trehalose dimycolate]). Test sera from the mice for antibody production after two booster immunisations. For the final immunisation, use WSSV structural proteins in the range of 15-35 kDa that have been eluted from SDS-PAGE gel slices and emulsified in synthetic adjuvant. Two days after immunisation, remove spleen cells from one mouse and fuse them with SP2/0-Ag-14 myeloma cells using polyethylene glycol. Use purified WSSV in a dot-immunoblot assay to screen the hybridoma for the production of WSSV-specific MAbs. The highly specific MAbs can be adapted to various test formats (49, 50). The following three formats are relatively rapid and sensitive enough and for the detection of WSSV in clinical shrimp specimens (49).
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| | | For fluorescent antibody detection of WSSV in fresh tissue impression smears, remove the stomachs of moribund shrimp and prepare impression smears on positively charged microscope slides. Make a sagittal section and transfer the epithelial cells to the slide. Immediately spray-fix using Surgipath cytology fixative and store at room temperature until used for reaction with the antibodies. Just before antibody staining, fix the smears by immersion in 100% methanol for 10 minutes Rehydrate the smears by incubation in three changes of PBS for 2 minutes Apply 500 µl of blocking reagent (PBS with 10% normal goat serum and 0.2% Hammersten casein) to the smears for 15 minutes at room temperature. Apply the MAbs for 45 minutes at room temperature. Wash the smears in three changes of PBS, and apply a fluorescein-conjugated goat anti-mouse IgG secondary antibody for 45 minutes at room temperature. Wash three times in PBS, and mount the smears with a cover-slip using a fluorescent mounting medium. Observe under an epifluorescent microscope. Green reactions are fluorescing WSSV-infected cells.
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| | | For immunohistochemistry on fixed sections, fix moribund shrimp with Davidson's AFA fixative for 24-48 hours. Embed the tissues in paraffin and cut into 4 µm sections. Place sections on to Superfrost Plus positively charged microscope slides. Heat at 65°C to melt the paraffin, rehydrate the sections through a series of washes in Hemo-De (a xylene substitute), 95% alcohol, 80% alcohol, 50% alcohol and distilled water. Incubate the sections for 5 minutes in PBS and then block with PBS containing 10% normal goat serum and 2% Hammersten casein for 15 minutes at room temperature. Apply the MAbs to the sections for 30 minutes at room temperature. Wash in three changes of PBS, and react the sections with a goat anti-mouse IgG (H& L) F(ab')2 antibody conjugated to alkaline phosphatase and diluted 1/1000 in PBS for 30 minutes at room temperature. Develop the reactions for 15-30 minutes using nitroblue tetrazolium and bromochloroindoyl phosphate in a pH 9.5 buffer. Counterstain the sections with Bismarck brown and dehydrate through a series of alcohol washes ending with Hemo-De. Mount with a cover-slip and permanent mounting medium and examine under a light microscope for the presence of a blue-black precipitate.
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| | | For the whole mount assay, fix tissue from WSSV-infected shrimp in Davidson's AFA and transfer to 70% alcohol after 24-48 hours. Cut the tissue into 2-4 mm blocks using a razor blade and place into a microfuge tube. All subsequent reactions are performed by adding 250-500 µl of reagent to the tube, allowing to incubate and then removing the solution with a pipette. First rehydrate the tissue by immersing in 50% ethanol for 15 minutes and wash six times with distilled water over a 10-minute period. Homogenise the tissue blocks with a plastic pestle and treat with a blocking reagent (PBS, 2% Hammersten casein, 10% normal goat serum) for 15 minutes at 37°C. React the tissues with 10 x concentrated MAb tissue culture supernatant fluid for 30 minutes at 37°C. Wash the tissues with several changes of PBS and add the secondary antibody (goat anti-mouse IgG [H& L] F[ab']2 conjugated to alkaline phosphatase and diluted 1/1000) for 30 minutes at 37°C. Wash the tissue again with PBS and add the detection reagent (nitroblue tetrazolium [NBT] and bromochloro-indoyl phosphate in pH 9.5 buffer) for 30-60 minutes at room temperature. Wash the tissue with distilled water, counterstain with Bismarck brown and dehydrate through a series of alcohol washes ending with Hemo-De. Place the crushed tissue on to a microscope slide, mount with a cover-slip and permanent mounting medium and examine under a light microscope for the presence of a blue-black precipitate within the nuclei of WSSV-infected cells.
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| | 1.4. | In-situ DNA hybridisation
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| | | The protocol described here was developed based on Nunan & Lightner (46), although an alternative protocol has been described by Chang et al. (2). In brief, place 5 µm paraffin sections on silanised or positively charged slides, and heat the slide on a hot plate at 65°C for 30 minutes. Deparaffinise, rehydrate and then treat for 2-30 minutes (depending on tissue type) with 100 µg/ml proteinase K in Tris/NaCl/EDTA (TNE) buffer at 37°C. Post-fix slides by chilling in pre-cooled 0.4% formaldehyde for 5 minutes at 4°C and wash the slides in 2 x standard saline citrate (SSC; 1 x SSC = 150 mM NaCl, 15 mM tri-sodium citrate, pH 7.0) at room temperature. Prehybridise the slides with prehybridisation solution (50% formamide, 0.2% Ficoll 400, 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, 5 x SSC, 1 mM EDTA, 50 mM Tris/HCl, pH 8) for 30 minutes at 42°C. Follow with hybridisation with the 1447 bp WSSV-specific PCR amplicon (described in Section 1.1. above) that has been labelled with digoxigenin (DIG), and then with visualisation using a light microscope. For hybridisation, boil the probe for 10 minutes and immediately place on ice. Dilute the probe to 30-50 ng/ml in prehybridisation solution and apply 500 µl to each slide. Put the slide on a hotplate at 85-95°C for 6-10 minutes (make sure that it does not reach boiling point), quench slides on ice for 5 minutes and then transfer to a humid chamber for 16-20 hours at 42°C. After hybridisation, wash the slides twice for 15 minutes each time with 2 x SSC at room temperature, twice for 5 minutes with 1 x SSC at 37°C and twice for 5 minutes with 0.5 x SSC at 37°C. For hybridisation detection, wash slides with maleic acid buffer (100 mM maleic acid, 150 mM NaCl, pH 7.5) for 5 minutes at room temperature. Block the slides with blocking solution (2% normal goat serum and 0.3% Triton X-100 in maleic acid buffer) for 30 minutes at 37°C. Add 250 µl anti-DIG alkaline phosphatase (AP)-conjugated antibody solution (1 µl/ml anti-DIG/AP-Fab fragment in maleic acid buffer containing 1% normal goat serum and 0.3% Triton X-100) to each slide, and incubate at 37°C for 30 minutes Wash the slides twice with maleic acid buffer for 10 minutes each and once with detection buffer (100 mM Tris/HCl, 100 mM NaCl, pH 9.5) at room temperature. Add 500 µl of development solution (prepare immediately before use by adding 45 µl NBT salt solution [75 mg/ml in 70% dimethyformamide], 35 µl 5-bromo-4-chloro-3-indoyl phosphate, toluidinum salt [X-phosphate] solution [50 mg/ml in dimethylformamide] and 1 ml 10% PVA to 9 ml of detection buffer) to each slide and incubate in the dark in a humid chamber for 1-3 hours. Stop the reaction by washing slides in TE buffer (10 mM Tri-HCl, 1 mM EDTA, pH 8.0) for 15 minutes at room temperature. Wash slides in distilled water for ten dips, counterstain the slides in 0.5% aqueous Bismarck Brown Y for 30-90 seconds and then rinse with water. Wet mount using aqueous mounting media for observation immediately or dehydrate the slides and mount with Permount mounting media for long-term preservation. Mount the slides with cover-slips and examine with a bright field microscope. Positive hybridisation appears as a dark blue to black precipitate against the yellow to brown counterstain.
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| 2. | Presumptive Diagnostic Methods for WSSV
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| | Presumptive diagnosis of WSSV can be achieved by the following method.
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| | 2.1. | Rapid staining of squash mount preparations
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| | | There are two general types of rapid squash mount preparations that can be used for presumptive diagnosis of white spot disease (WSD). One employs fresh, unstained wet mounts fixed with formalin and viewed by dark-field microscopy with a wet-type condenser. The other employs fixed tissue stained with H& E.
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| | | The dark-field technique is described by Momoyama et al. (42). In brief: dissect out the stomach as a source of subcuticular tissue or peel off thin layers of subcuticular tissue from the cephalothorax and fix in a 10% formalin solution. Using fine forceps spread thin pieces of the subcuticular tissue on a slide in a small volume of 10% formalin. Add a cover-slip and remove excess solution by placing a filter paper at the edge of the cover-slip. Using dark-field optics, focus the microscope on an area of the preparation where shrimp pigment cells are poorly distributed. Specimens with WSD will show moderate to large numbers of refractile, hypertrophied nuclei.
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| | | For rapidly stained H& E preparations (10), collect moribund shrimp from a suspected outbreak of WSD and fix the whole shrimp or gill filaments in Davidson's fixative (26) overnight. After fixation, wash some gill filaments thoroughly with tap water to remove the fixative. Then stain with Meyer's H& E (26). After staining and dehydration, when the tissue is in xylene, place a gill filament on a microscope slide in a drop of xylene. Using a fine pair of needles (a stereo microscope is helpful), break off several secondary filaments and then replace the main filament in xylene where it can be stored indefinitely as a permanent reference in a sealed vial. Being careful not to let the xylene dry, tease apart the secondary filaments on the microscope slide and remove any large fragments or particles that would thicken the mount unnecessarily. Finally, add a drop of mounting fluid and a cover glass. Use light pressure to flatten the mount as much as possible. This procedure may also be used with thin layers of subcuticular tissue. With WSD outbreaks, examination with the x40 objective of the light microscope will reveal the presence of moderate to large numbers of hypertrophied nuclei with basophilic central inclusions surrounded by marginated chromatin (10). It is important also to detect some nuclei with Cowdry type-A inclusions characteristic of the early stage of WSSV infection. Like the fixed tissues and the filaments in xylene, these whole mount slides can be kept as a permanent record.
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| | | In the event that rapid results are required, the fixation step can be shortened to only 2 hours by changing the acetic acid portion in the Davidson's fixative formula to 50% concentrated HCl (10). For best results, this fixative should not be stored for more than a few days before use. After fixation, wash thoroughly to remove the fixative and check that the pH has returned to near neutral before staining. Do not fix for longer periods or above 25°C as this may result in excessive tissue damage that will make interpretation difficult or impossible.
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| | 2.2. | Bioassay
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| | | Bioassay methods for WSSV are available, but their application is limited to labs with access to susceptible WSSV-free reference shrimp stocks (8, 47). For bioassay, remove the pleopods from shrimp suspected of WSSV infection and homogenise in TN buffer (0.02 M Tris/HCl, 0.4 M NaCl, pH 7.4). Following centrifugation at 1000 g for 10 minutes, dilute the supernatant fluid 1/10 with 2% NaCl and filter (0.2 µm filter). Inject 0.2 ml of inoculum into the dorso-lateral aspect of the fourth abdominal segment of indicator shrimp (specific pathogen free shrimp at juvenile stage), injecting between the tergal plates into the muscle of the third abdominal segment. Examine moribund shrimp grossly orby using the methods described in Section 2.1.
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| 3. | Confirmatory Diagnostic Methods for WSSV
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| | Confirmatory diagnosis of WSSV can be achieved by any of the following methods.
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| | 3.1. | Histopathology of tissue sections
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| | | Fix moribund shrimp from a suspected WSD outbreak in Davidson's fixative and process for preparation of standard H& E-stained tissue sections (1, 26). Examine the sections by light microscopy for the presence of moderate to large numbers of hypertrophied nuclei with basophilic central inclusions surrounded by marginated chromatin in tissues of ectodermal and mesodermal origin (66). Some of the hypertrophied nuclei will have Cowdry type-A inclusions. Particularly useful for viewing WSD histopathology are the subcuticular tissues of the stomach and cephalothorax and the gills.
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| | 3.2. | Nested PCR of tissues and haemolymph (see Section 1.1.)
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| | 3.3. | Antibody-based assays (see Section 1.3.)
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| | 3.4. | In-situ DNA hybridisation (see Section 1.4.)
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| | 3.5. | Transmission electron microscopy
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| | | For TEM, the most suitable tissues of moribund shrimp suspected of WSV infection are subcuticular tissues, gills and pereiopods. For screening or surveillance of grossly normal shrimp, the most suitable tissue is subcuticular tissue from the stomach. The reagents described below are from ref. 24. In brief, stun living shrimp by immersion in ice water until just immobilised or kill by injection of fixative. Quickly dissect and remove small portions of target tissue (no larger than a few millimetres in diameter) and fix in at least 10 volumes of 6% glutaraldehyde held at 4°C and buffered with sodium cacodylate (Na[CH3]2AsO2.3H2O) solution (Na cacodylate 8.6 g, NaCl 10 g, distilled water to make 100 ml adjusted to pH 7 with 0.2 N HCl) or phosphate solution (NaH2PO4.H2O 0.6 g, Na2HPO4 1.5g, NaCl 1 g, sucrose 0.5 g, distilled water to make 100 ml adjusted to pH 7 with 0.2 N HCl). Fix for at least 24 hours prior to processing. For long-term storage in fixative at 4°C, reduce glutaraldehyde to 0.5~1.0%. Processing involves post-fixation with 1% osmium tetroxide, dehydration, embedding, sectioning and staining with uranyl acetate and lead citrate according to standard TEM methods.
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